US7998940B2 - Aptamers to von Willebrand factor and their use as thrombotic disease therapeutics - Google Patents

Aptamers to von Willebrand factor and their use as thrombotic disease therapeutics Download PDF

Info

Publication number
US7998940B2
US7998940B2 US12/322,078 US32207809A US7998940B2 US 7998940 B2 US7998940 B2 US 7998940B2 US 32207809 A US32207809 A US 32207809A US 7998940 B2 US7998940 B2 US 7998940B2
Authority
US
United States
Prior art keywords
seq
aptamer
von willebrand
ome
willebrand factor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related, expires
Application number
US12/322,078
Other languages
English (en)
Other versions
US20090149643A1 (en
Inventor
John L. Diener
H. A. Daniel Lagasse
Claude Benedict
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Archemix Corp
Original Assignee
Archemix Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US11/222,346 external-priority patent/US7566701B2/en
Application filed by Archemix Corp filed Critical Archemix Corp
Priority to US12/322,078 priority Critical patent/US7998940B2/en
Publication of US20090149643A1 publication Critical patent/US20090149643A1/en
Assigned to ARCHEMIX CORP. reassignment ARCHEMIX CORP. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LAGASSE, H.A. DANIEL, DIENER, JOHN L., BENEDICT, CLAUDE
Application granted granted Critical
Publication of US7998940B2 publication Critical patent/US7998940B2/en
Expired - Fee Related legal-status Critical Current
Adjusted expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/317—Chemical structure of the backbone with an inverted bond, e.g. a cap structure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/352—Nature of the modification linked to the nucleic acid via a carbon atom
    • C12N2310/3521—Methyl

Definitions

  • the invention relates generally to the field of nucleic acids and more particularly to aptamers capable of binding to von Willebrand Factor useful as therapeutics in and diagnostics of thrombotic diseases and/or other diseases or disorders in which von Willebrand Factor mediated platelet aggregation is implicated.
  • the invention further relates to materials and methods for the administration of aptamers capable of binding to von Willebrand Factor.
  • Aptamers are nucleic acid molecules having specific binding affinity to molecules through interactions other than classic Watson-Crick base pairing.
  • Aptamers like peptides generated by phage display or monoclonal antibodies (“mAbs”), are capable of specifically binding to selected targets and modulating the target's activity or binding interactions, e.g., through binding aptamers may block their target's ability to function. Discovered by an in vitro selection process from pools of random sequence oligonucleotides, aptamers have been generated for over 130 proteins including growth factors, transcription factors, enzymes, immunoglobulins, and receptors.
  • mAbs monoclonal antibodies
  • a typical aptamer is 10-15 kDa in size (20-45 nucleotides), binds its target with nanomolar to sub-nanomolar affinity, and discriminates against closely related targets (e.g., aptamers will typically not bind other proteins from the same gene family).
  • a series of structural studies have shown that aptamers are capable of using the same types of binding interactions (e.g., hydrogen bonding, electrostatic complementarities, hydrophobic contacts, steric exclusion) that drive affinity and specificity in antibody-antigen complexes.
  • Aptamers have a number of desirable characteristics for use as therapeutics and diagnostics including high specificity and affinity, biological efficacy, and excellent pharmacokinetic properties. In addition, they offer specific competitive advantages over antibodies and other protein biologics, for example:
  • aptamers can be administered by subcutaneous injection (aptamer bioavailability via subcutaneous administration is >80% in monkey studies (Tucker et al., J. Chromatography B. 732: 203-212, 1999)). With good solubility (>150 mg/mL) and comparatively low molecular weight (aptamer: 10-50 kDa; antibody: 150 kDa), a weekly dose of aptamer may be delivered by injection in a volume of less than 0.5 mL.
  • the small size of aptamers allows them to penetrate into areas of conformational constrictions that do not allow for antibodies or antibody fragments to penetrate, presenting yet another advantage of aptamer-based therapeutics or prophylaxis.
  • aptamers are chemically synthesized and consequently can be readily scaled as needed to meet production demand. Whereas difficulties in scaling production are currently limiting the availability of some biologics and the capital cost of a large-scale protein production plant is enormous, a single large-scale oligonucleotide synthesizer can produce upwards of 100 kg/year and requires a relatively modest initial investment.
  • the current cost of goods for aptamer synthesis at the kilogram scale is estimated at $500/g, comparable to that for highly optimized antibodies. Continuing improvements in process development are expected to lower the cost of goods to ⁇ $100/g in five years.
  • Therapeutic aptamers are chemically robust. They are intrinsically adapted to regain activity following exposure to factors such as heat and denaturants and can be stored for extended periods (>1 yr) at room temperature as lyophilized powders.
  • vWF The von Willebrand Factor
  • vWF is a mediator of platelet adhesion at sites of vascular damage.
  • vWF is a large multi-subunit, multimeric soluble factor mainly produced by vascular endothelial cells.
  • the von Willebrand Factor becomes immobilized on the blood vessel wall via interactions between von Willebrand Factor domain A3 and exposed collagen.
  • GPIb platelet-receptor glycoprotein Ib
  • A1 domain of the immobilized von Willebrand Factor facilitates the adhesion and activation of platelets at sites of vascular injury.
  • GPIb platelet-receptor glycoprotein Ib
  • the von Willebrand factor is pro-thrombotic, playing an important role during hemostasis in facilitating thrombus formation at sites of vascular injury.
  • the von Willebrand Factor by the same mechanism, also plays a key role in pathological conditions, such as cardiovascular diseases, involving platelet aggregation and thrombosis formation.
  • cardiovascular diseases involving platelet aggregation and thrombosis formation.
  • antithrombotic therapies are currently available there is still a large unmet need for additional therapies.
  • the American Heart Association estimates that more than 60 million people in the United States alone have one or more forms of cardiovascular disease, and that a high proportion of people with cardiovascular disease are at higher risk for arterial thrombosis. S. P. Jackson and S. M. Schoenwaelder, Nature Reviews, 2, 1-12 (2003).
  • a significant problem with presently available therapies is that improving efficacy reduces safety. S. P. Jackson and S. M. Schoenwaelder, Nature Reviews, 2, 1-12 (2003). Accordingly, it would be beneficial to treat or prevent thrombotic disease by preventing platelet aggregation in the vasculature while minimizing bleeding side effects.
  • the present invention provides materials and methods to meet these and other needs.
  • FIG. 1 is a schematic representation of the in vitro aptamer selection (SELEXTM) process from pools of random sequence oligonucleotides.
  • FIG. 2 is an illustration depicting various PEGylation strategies representing standard mono-PEGylation, multiple PEGylation, and oligomerization via PEGylation.
  • FIG. 3 is a table listing the amino acid sequences of the von Willebrand Factor domain A1 proteins used in the experiments of the invention.
  • FIG. 4 is a table listing the amino acid sequence of the full length human von Willebrand Factor protein used in the experiments of the invention.
  • FIG. 5 is an illustration depicting the proposed secondary structure of ARC1029 (SEQ ID NO 214).
  • FIG. 6 is a graph of the dot blot binding curves for ARC1029 (SEQ ID NO 214) to full length vWF and rabbit vWF domain A1.
  • a black box in this table indicates a deletion.
  • FIG. 7 is a graph of FACS data showing that ARC1029 (SEQ ID NO 214) inhibits binding of human vWF and rabbit vWF A1 domain to lyophilized human platelets.
  • FIG. 8 is a graph of an aggregometer trace showing ARC1029 (SEQ ID NO 214) inhibiting botrocetin induced platelet aggregation over time.
  • FIG. 9 is a graphical representation of ARC1029 (SEQ ID NO 214) inhibiting botrocetin induced platelet aggregation.
  • FIG. 11 is an illustration depicting the sequence alignment for three aptamers of the vWF rRdY SELEXTM Family #1.
  • FIG. 12 is an illustration depicting the proposed secondary structure for SEQ ID NO 218.
  • FIG. 13 is an illustration depicting the proposed secondary structure for SEQ ID NO 219.
  • FIG. 14 is an illustration depicting the sequence alignment for 26 aptamers of the vWF DNA SELEXTM 2 Family #1.
  • the black line at the top of the alignment represents the proposed core nucleic acid binding sequence required to bind the von Willebrand Factor target.
  • FIG. 15A is an illustration depicting the proposed secondary structure for SEQ ID NO 220.
  • FIG. 15B is an illustration depicting the secondary structure of ARC1172 (SEQ ID NO 222) and which residues are tolerant of 2′-OMe substitution.
  • FIG. 16 is a table depicting the nucleic acid sequences including any modifications of ARC1029 (SEQ ID NO 214), ARC1115 (SEQ ID NO 221), ARC1172 (SEQ ID NO 222), and ARC1194 (SEQ ID NO 223) to ARC1243 (SEQ ID NO 272).
  • FIG. 17 is a table depicting the nucleic acid sequences including any modifications of ARC1172 (SEQ ID NO 222), ARC1338 (SEQ ID NO 273) to ARC1348 (SEQ ID NO 283) and ARC1361 (SEQ ID NO 284) to ARC1381 (SEQ ID NO 304).
  • a black block in this table indicates a deletion.
  • An “s” preceding a nucleotide indicator e.g. T or dA indicates a phosphorothioate substitution in the phosphate backbone 5′ to the indicated nucleotide.
  • FIG. 18 is a table depicting the nucleic acid sequences including any modifications of ARC1172 (SEQ ID NO 222), ARC1524 (SEQ ID NO 305) to ARC1535 (SEQ ID NO 316), ARC1546 (SEQ ID NO 317) and ARC1759 (SEQ ID NO 318).
  • a black block in this table indicates a deletion.
  • FIG. 19 is an illustration of the secondary structures of ARC1368 (SEQ ID NO 291) and ARC1534 (SEQ ID NO 315).
  • FIG. 20 is a graph depicting the clotting time, in the PFA-100 assay, for human whole blood treated with ARC1368 (SEQ ID NO 291) or ARC1525 (SEQ ID NO 306) as a function of aptamer concentration.
  • FIG. 21 is a graph depicting occlusion time, in a PFA-100 assay, of human whole blood treated with IntegrilinTM, ReoPrOTM or ARC1368 (SEQ ID NO 291), as a function of drug concentration.
  • FIG. 22 is a graph depicting percent inhibition, in BIPA, of human PRP, treated with IntegrilinTM, ReoPrOTM or ARC1368 (SEQ ID NO 291), as a function of drug concentration.
  • FIG. 23 is a graph depicting percent inhibition, in AIPA, of human PRP, treated with IntegrilinTM, ReoProTM or ARC1368 (SEQ ID NO 291), as a function of drug concentration.
  • FIG. 24 is a graph depicting percentage of full length ARC1172 (SEQ ID NO 222) or ARC1368 (SEQ ID NO 291), detected in human plasma, as a function of time.
  • FIG. 25 is a graph depicting primate plasma aptamer concentration (determined using Oligreen analysis) plotted as a function of time following administration of ARC1368 (SEQ ID NO 291), ARC1779 (SEQ ID NO 320) or ARC1780 (SEQ ID NO 321).
  • FIG. 26 is a graph showing the time points on the horizontal axis at which blood for testing was drawn from three cynomolgus macaques, ARC1779 (SEQ ID NO 320) plasma concentration (in nM) along the top third of the vertical axis, PFA-100 closure time (in seconds) on the middle third of the vertical axis, and the template or cutaneous bleeding time (in minutes) on the bottom third of the vertical axis.
  • the average from all three animals for plasma aptamer concentration, PFA-100 closure time and cutaneous bleeding time is plotted on the top third, middle third and bottom third of the graph, respectively.
  • FIG. 27 is a table showing the cutaneous bleeding time (CBT) in minutes, raw BIPA data and PFA-100 closure time (sec) at various time points, shown in column 1, relative to ARC1779 (SEQ ID NO 320) dosing in three different cynomolgus macaques.
  • FIG. 28 is a graph showing the average PFA-100 closure time at various time points following ARC1779 (SEQ ID NO 320) dosing of C. macaques.
  • FIG. 29 is a graph showing the average bleeding time of the three ARC1779 (SEQ ID NO 320) treated macaques taken at various time points following dosing.
  • FIG. 30 is a graph correlating the average bleeding time in ARC1779 (SEQ ID NO 320) treated C. macaques (left vertical axis) to the PFA-100 closure time.
  • FIG. 31 is a schematic depicting the blood sample collection schedule used in the assessment of ARC1779 (SEQ ID NO 320) in the cynomolgus monkey electrolytic thrombosis model.
  • FIG. 32 is graph of ARC1779 (SEQ ID NO 320) plasma concentration (vertical axis) as a function of time in each cynomolgus monkey of treatment group 3 tested in the electrolytic thrombosis model.
  • FIG. 33 is a graph of the time to occlusion of the right (hatched bar) or left carotid artery (indicated by a solid bar) in each cynomolgus macaque from each treatment group tested in the cynomolgus monkey electrolytic thrombosis model.
  • Bar pairs 1, 8 and 9 indicate treatment group 1 (vehicle only)
  • Bar pairs 2, 10, 11, 12 and 13 indicates treatment groups 2 and 4 (ReoPro).
  • Bar pairs 3 to 7 indicate treatment group 3 (1000 nM aptamer plasma concentration target group).
  • Bar pairs 20, 22, 16, and 18 indicate treatment group 7 (750 nM plasma aptamer concentration target group).
  • Bar pairs 19, 21, 23 and 24 indicate treatment group 6 (500 nM plasma aptamer concentration target group).
  • Bar pairs 15 and 17 indicate treatment group 5 (300 nM plasma aptamer concentration target group)
  • FIG. 34 is a graph showing the cutaneous bleed time in minutes (vertical axis) of the various cynomolgous treatment groups in the electrical injury model taken at the time points shown on the horizontal axis.
  • the present invention provides materials and methods for the treatment of thrombotic disorders involving von Willebrand Factor mediated platelet aggregation.
  • the present invention provides aptamers that specifically bind to a von Willebrand Factor target.
  • the von Willebrand Factor target is human von Willebrand Factor.
  • the von Willebrand Factor target is a variant of human von Willebrand Factor that performs a biological function that is essentially the same as a function of human von Willebrand Factor.
  • the biological function of the von Willebrand Factor target or variant thereof is to mediate platelet aggregation.
  • the variant of the human von Willebrand Factor target has substantially the same structure and substantially the same ability to bind an aptamer of the invention as that of human von Willebrand Factor.
  • the vWF target is a non-human von Willebrand Factor.
  • the aptamer of the invention binds the von Willebrand Factor target or a variant thereof that comprises an amino acid sequence which is at least 75%, 80%, 90% or 95% identical to SEQ ID NO 7 ( FIG. 4 ).
  • the von Willebrand Factor target comprises the amino acid sequence of SEQ ID NO 7.
  • sequence identity or “% identity” in the context of two or more nucleic acid or protein sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • sequence comparison algorithm test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Ausubel, F. M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987).
  • BLAST basic local alignment search tool
  • NCBI National Center for Biotechnology Information
  • sequence identity is determined using the BLAST implemented algorithm of Altschul et al., and the default parameters of McGinnis et al., which herein may be referred to as BLAST percent identity.
  • the vWF aptamer of the invention comprises a dissociation constant for human von Willebrand Factor or a variant thereof, of about 100 nM or less, preferably 50 nM or less, preferably 10 nM or less, preferably 5 nM or less, preferably 1 nM or less, and more preferably 500 pM or less.
  • the present invention also provides an aptamer that specifically binds to a von Willebrand Factor domain A1 target.
  • the von Willebrand Factor domain A1 target is a human von Willebrand Factor domain A1 target.
  • the human von Willebrand Factor domain A1 target is a variant of human von Willebrand Factor domain A1 that performs a biological function that is essentially the same as a function of human von Willebrand Factor domain A1.
  • the biological function of von Willebrand Factor domain A1 or a variant thereof is to bind to platelets.
  • the variant of human von Willebrand Factor domain target has substantially the same structure and substantially the same ability to bind said aptamer as that of human von Willebrand Factor domain A1.
  • the von Willebrand Factor domain A1 target is non-human von Willebrand Factor domain A1 target, e.g. a rabbit or non-human primate von Willebrand Factor domain A1 target.
  • the von Willebrand Factor domain A1 target of the invention comprises an amino acid sequence which is at least 75%, 80%, 90% or 95% identical to any one of the sequences selected from the group consisting of: SEQ ID NOS 4 to 6.
  • the von Willebrand Factor domain A1 target comprises any one of the amino acid sequences selected from the group consisting: of SEQ ID NOS 4 to 6.
  • the vWF aptamer of the invention comprises a dissociation constant for human von Willebrand Factor domain A1 or a variant thereof, of about 100 nM or less, preferably 50 nM or less, preferably 10 nM or less, preferably 5 nM or less, preferably 1 nM or less, and more preferably 500 pM or less.
  • the aptamer of the invention comprises a dissociation constant for non-human von Willebrand Factor domain A1 or a variant thereof, of about 100 nM or less, preferably 50 nM or less, preferably 10 nM or less, preferably 5 nM or less, preferably 1 nM or less, and more preferably 500 pM or less.
  • the invention provides an aptamer that specifically binds to a von Willebrand Factor full length target. In some embodiments, the invention provides an aptamer that specifically binds to a von Willebrand Factor full length target and a von Willebrand Factor domain A1 target. In some embodiments, the von Willebrand Factor full length target is a human von Willebrand target or variant thereof. In other embodiments, the von Willebrand Factor full length target is a non-human von Willebrand target or variant thereof. In some embodiments, the von Willebrand Factor domain A1 target is a non-human von Willebrand Factor domain A1 target or variant thereof.
  • the von Willebrand Factor domain A1 target is a human von Willebrand Factor domain A1 target or variant thereof.
  • the von Willebrand Factor full length target or domain A1 target is selected from the group consisting of: a rabbit, guinea pig, monkey, dog, sheep, mouse and rat, von Willebrand Factor full length or domain A1 target.
  • the von Willebrand Factor full length target and von Willebrand Factor domain A1 target to which the aptamer of the invention specifically binds are from different species.
  • the present invention provides aptamers against a von Willebrand Factor target that are ribonucleic acid or deoxyribonucleic acid or mixed ribonucleic acid and deoxyribonucleic acid.
  • Aptamers of the invention may be single stranded ribonucleic acid or deoxyribonucleic acid or mixed ribonucleic acid and deoxyribonucleic acid.
  • the aptamer of the invention comprises at least one chemical modification.
  • the chemical modification is selected from the group consisting of: a chemical substitution at a sugar position; a chemical substitution at a phosphate position; and a chemical substitution at a base position, of the nucleic acid.
  • the chemical modification is selected from the group consisting of: incorporation of a modified nucleotide, 3′ capping, conjugation to a high molecular weight, non-immunogenic compound, conjugation to a lipophilic compound, and incorporation of phosphorothioate into the phosphate back bone.
  • the non-immunogenic, high molecular weight compound is polyalkylene glycol, more preferably polyethylene glycol.
  • an aptamer e.g. a von Willebrand Factor aptamer, that binds specifically to a target wherein the aptamer comprises a nucleotide sequence having no more than four, no more than three, no more than two or no more than one phosphorothioate backbone modifications, and the aptamer has a binding affinity for the target wherein the binding affinity is increased relative to a second aptamer having the same nucleotide sequence but lacking phosphorothioate back bone modification is provided.
  • the target is a protein or peptide not having the known function of binding nucleic acid, particularly not have the known primary function of binding nucleic acid.
  • the aptamer of the invention modulates a function of any one of the group consisting of: the von Willebrand Factor target, the von Willebrand Factor domain A1 target and a variant of either target.
  • the modulated function is platelet aggregation mediation.
  • the aptamer of the invention inhibits von Willebrand Factor mediated platelet aggregation in vivo.
  • the aptamer of the invention prevents binding of any one of the group consisting of: the von Willebrand Factor target, the von Willebrand Factor domain A1 target and a variant thereof, to a platelet.
  • the aptamer of the invention prevents binding of any one of the group consisting of: the von Willebrand Factor target, the von Willebrand Factor domain A1 target and a variant of either target, to a platelet receptor protein. In yet other embodiments, the aptamer of the invention prevents binding of any one of the group consisting of: the von Willebrand Factor target, the von Willebrand Factor domain A1 target and a variant of either target, to the platelet receptor protein GPIb. In some embodiments, the aptamer of the invention prevents vWF Factor mediated platelet aggregation while not significantly increasing bleeding time.
  • a non-significant increase in bleeding time is less than 15, minutes, preferably less than 10 minutes, more preferably less than 5 minutes, and in some embodiments, less than 3 minutes relative to the bleeding time of a subject not treated with the aptamer of the invention.
  • the bleeding time is determined by cutaneous (or template) bleeding time.
  • the aptamer of the invention has substantially the same ability to bind any one of the group consisting of: the von Willebrand Factor target, the von Willebrand Factor domain A1 target and a variant thereof, as that of an aptamer selected from the group consisting of: SEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98 to 164, SEQ ID NO 165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS 212-214, ARC1115 (SEQ ID NO 221), ARC1172 (SEQ ID NO 222), ARC1194 (SEQ ID NO 223) to ARC1240 (SEQ ID NO 269)
  • the aptamer of the invention has substantially the same structure and substantially the same ability to bind of any one of the group consisting of: the von Willebrand Factor target, the von Willebrand Factor domain A1 target and a variant thereof, as that of an aptamer selected from the group of sequences consisting of: SEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98 to 164, SEQ ID NO 165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS 212-214, ARC1115 (SEQ ID NO 221), ARC1172 (SEQ ID NO 222), ARC1194 (SEQ ID NO 223) to ARC
  • the aptamer of the invention is selected from the group consisting of: SEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98 to 164, SEQ ID NO 165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS 212-214, ARC1115 (SEQ ID NO 221), ARC1172 (SEQ ID NO 222), ARC1194 (SEQ ID NO 223) to ARC1240 (SEQ ID NO 269), ARC1338 (SEQ ID NO 273) to ARC1346 (SEQ ID NO 281), ARC1361 (SEQ ID NO 284) to ARC1381 (SEQ ID NO 304),
  • the aptamer of the invention comprises the primary nucleic acid sequence of ARC1172 (SEQ ID NO 222) or ARC11115 (SEQ ID NO 221) or ARC1029 (SEQ ID NO 214) or SEQ ID NO 220 and does not comprise a 2′-O-Me substituted nucleotide at position 6 to 9, 20, 22, 24 to 27, 30 or 32 to 33.
  • the aptamer of the invention comprises the nucleic acid sequence of ARC1172 (SEQ ID NO 222) or ARC1115 (SEQ ID NO 221) or ARC1029 (SEQ ID NO 214) or SEQ ID NO 220 and comprises a 2′-O-Me substituted nucleotide at one or more positions, at 5 or more positions, at 10 or more positions, at 15 or more positions, or at 20 or more positions.
  • the aptamer of the invention comprises the nucleic acid sequence of ARC1172 (SEQ ID NO 222) or ARC1115 (SEQ ID NO 221) or ARC1029 (SEQ ID NO 214) or SEQ ID NO 220 and comprises a 2′-O-Me substituted nucleotide at all positions selected from the group consisting of: position 1 to 5, position 10 to 19, position 21, position 23, position 28 to 29, and position 34 to 41 wherein the position numbering starts at the 5′ end of the nucleic acid sequence.
  • an aptamer of the invention is selected from the group consisting of: SEQ ID NO 217 and 220 wherein N x -N x -N x -N x -, or N (3-10) may be replaced with a PEG linker.
  • the aptamer that binds specifically to von Willebrand Factor comprises a three way helix junction secondary structure motif having the consensus sequence structure of SEQ ID NO 220 depicted in FIG. 15 .
  • the aptamer having the three way helix junction comprises the consensus structure depicted in FIG. 19 A (ARC1368 (SEQ ID NO 291)). While in another embodiment, the aptamer having the three way helix junction comprises the consensus structure depicted in FIG. 19 B (ARC1534 (SEQ ID NO 315)).
  • the aptamer that binds specifically to von Willebrand Factor comprises a stem-loop-stem-loop secondary structure motif having the consensus sequence structure of SEQ ID NO 217 depicted in FIG. 10 .
  • the aptamer that specifically binds to von Willebrand Factor comprises the stem-loop-loop secondary structure motif having the consensus sequence structure of SEQ ID NO 218 depicted in FIG. 12 .
  • the aptamer that binds specifically to von Willebrand Factor comprises a three way junction secondary structure motif with two helical stems and a stem-loop of SEQ ID NO 19 as depicted in FIG. 13 .
  • the secondary structure motif of the aptamer of the invention is predicted by: RNAstructure, Version 4.1 (Mathews, D. H.; Disney, M. D.; Childs, J. L.; Schroeder, S. J.; Zuker, M.; and Turner, D. H., “Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure,” 2004 . Proceedings of the National Academy of Sciences, US, 101, 7287-7292).
  • an aptamer that specifically binds to a human von Willebrand Factor target and to a non-human von Willebrand Factor target is provided.
  • an aptamer comprising the following structure or a salt thereof is provided:
  • the aptamer of the invention comprises the following structure:
  • the aptamer nucleic acid sequence is an anti-vWF aptamer of the invention.
  • the aptamer comprises the following nucleic acid sequence or fragment thereof: mGmCmGmUdGdCdAmGmUmGmCmCmUmUmCmGmGmCdCmG-s-dTmGdCdGdGTmGmCdCmUdCdCmGmUdCmAmCmGmC-3T (SEQ ID NO 291) wherein m refers to a 2′-OMe substitution, the “d” refers to a deoxy nucleotide, the “s” refers to a phosphorothioate substitution and “3T” refers to an inverted deoxy thymidine.
  • the aptamer comprises the following nucleic acid sequence or fragment thereof: dGdGdCdGdTdGdCdAdGdTdGdCdCdTdTdCdGdGdCdTdGdGdTdGdCdGdTdGdCdGdTdGdCdTdGdCdTdCdC dGdTdCdC dGdTdCdC dCdC dGdTdCdC dGdTdCdC dGdTdCdC dGdTdCdC dGdTdCdAdCdGdCdC-3T (SEQ ID NO 323) wherein “d” refers to a deoxy nucleotide and “3T” refers to an inverted deoxy thymidine.
  • a salt of an aptamer of the invention is provided.
  • the following salt of an aptamer is provided:
  • a pharmaceutical composition comprising a therapeutically effective amount of any one of the aptamers of the invention or a salt thereof and a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutical composition of the invention comprises ARC1779.
  • the pharmaceutical composition comprises an aptamer having the following structure or a salt thereof:
  • the aptamer comprises the following nucleic acid sequence or fragment thereof: mGmCmGmUdGdCdAmGmUmGmCmCmUmUmCmGmGmCdCmG-s-dTmGdCdGdGTmGmCdCmUdCdCmGmUdCmAmCmGmC-3T (SEQ ID NO 291) wherein m refers to a 2′-OMe substitution, the “d” refers to a deoxy nucleotide, the “s” refers to a phosphorothioate substitution and “3T” refers to an inverted deoxy thymidine.
  • the invention provides a method of treating, preventing or ameliorating a disease mediated by von Willebrand Factor, comprising administering an aptamer or a pharmaceutical composition of the invention to a vertebrate, preferably a mammal, more preferably a human.
  • the disease to be treated, prevented or ameliorated is selected from the group consisting of: essential thrombocytopenia, thrombotic thrombocopenic purpura (“TTP”), Type IIb von Willebrand's disease, pseudo von Willebrand disease, peripheral artery disease, e.g.
  • peripheral arterial occlusive disease unstable angina, angina pectoris, arterial thrombosis, atherosclerosis, myocardial infarction, acute coronary syndrome, atrial fibrillation, carotid stenosis, cerebral infarction, cerebral thrombosis, ischemic stroke, and transient cerebral ischemic attack.
  • the pharmaceutical composition of the invention is administered prior to/during and/or after dialysis, CABG surgery, percutaneous coronary intervention or heart valve replacement.
  • the length of the in vivo half life of the aptamer of the invention may vary depending on the disease to be treated. ameliorated and/or prevented.
  • the aptamer of the invention may comprise a relatively long half life, e.g. a half life greater than five hours in humans.
  • the aptamer of the invention comprises a desired functional half life or duration of effect.
  • Functional half life or duration of effect is a function of both pharmacokinetic half life and pharmacodynamic activity of the aptamer.
  • the desired human functional half-life or duration of effect for an anti-vWF therapeutic aptamer is on the order of 1-5 hours for the proposed indications elective PCI and ACS.
  • Aptamers with such kinetics represent a balance between the dual objectives of (1) minimizing total aptamer dose (achieved with longer half-life) and (2) allowing rapid normalization of platelet function following cessation of treatment (achieved with shorter half-life).
  • rapid normalization of platelet function is important as it allows clinicians the option of rapid intervention (e.g. CABG) should a patient fail to stabilize in response to treatment.
  • the aptamer for use in the methods of treatment and/or pharmaceutical compositions of the invention comprises a relatively short functional half life, e.g. a functional half life in humans of about 1 to 5 hours.
  • the functional half life in humans is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours and not more than about 5 hours.
  • the functional half life or duration of effect is about the same as the distribution half life T 1/2 ⁇ of the aptamer.
  • the aptamer of the invention comprising the short functional half life in humans is for use in methods and compositions for the treatment, amelioration or prevention of diseases that potentially may require surgical intervention, such as acute coronary syndrome.
  • the aptamer of this aspect of the invention comprising a short functional half life in humans is conjugated to a PEG, e.g. a 5, 10 or 20 kDa PEG.
  • the aptamer of this aspect of the invention comprising a short half life in humans is ARC1779.
  • the invention also provides a diagnostic method comprising contacting an aptamer of the invention with a composition suspected of comprising von Willebrand Factor, von Willebrand Factor domain A1 or a variant thereof and detecting the presence or absence of von Willebrand Factor, von Willebrand Factor domain A1 or a variant thereof.
  • the diagnostic method is for use in vitro while in other embodiments, the diagnostic method is for use in vivo.
  • the invention also provides a method for identifying an aptamer that blocks a biological function in vivo comprising:
  • the identification method further comprises;
  • the identification method further comprises selecting an aptamer that blocks a biological function of the full length protein target in vivo while in other embodiments, the method further comprises selecting an aptamer that blocks a biological function of the protein target domain in vivo.
  • the full length protein target is from a first species and the protein target domain is from a second species.
  • the method further comprises selecting an aptamer capable of binding to both protein targets of both the first and second species, preferably selecting an aptamer that blocks a biological function of the protein target in both the first and second species.
  • the full length target protein target is von Willebrand Factor.
  • the full length target protein target is von Willebrand Factor, wherein it is preferred that the selected aptamer blocks von Willebrand Factor mediated platelet aggregation.
  • the protein target domain is von Willebrand Factor domain A1.
  • the invention also provides an aptamer identified by the identification method of the invention.
  • the invention also provides an aptamer that specifically binds to von Willebrand Factor comprising a primary nucleic acid sequence at least 80% identical, particularly at least 90% identical, and more particularly at least 95% identical to any one of the primary nucleic acid sequences selected from the group consisting of SEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98 to 164, SEQ ID NO 165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS 212-214, ARC1115 (SEQ ID NO 221), ARC1172 (SEQ ID NO 222), ARC1194 (SEQ ID NO 223) to ARC1240 (SEQ ID NOS 11 to
  • the aptamer of the invention comprises a nucleic acid sequence having chemical modifications that including chemical modifications is at least 80% identical, particularly 90% identical, and more particularly at least 95% identical to any one of the nucleic acid sequences selected from the group consisting of: SEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98 to 164, SEQ ID NO 165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS 212-214, ARC115 (SEQ ID NO 221), ARC1172 (SEQ ID NO 222), ARC1194 (SEQ ID NO 223) to ARC1240 (SEQ ID NO 269), ARC1338 (SEQ
  • the invention provides an aptamer that upon binding a von Willebrand Factor target modulates a von Willebrand Factor function, preferably in vivo and comprises a sequence of 30 contiguous nucleotides that are identical to a sequence of 30 contiguous nucleotides comprised in any one of the sequences selected from the group of: SEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98 to 164, SEQ ID NO 165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS 212-214, ARC1115 (SEQ ID NO 221), ARC1172 (SEQ ID NO 222), ARC1194 (SEQ ID NO
  • the aptamer of the invention upon binding a von Willebrand Factor target modulates a von Willebrand Factor function, preferably in vivo, and comprises 20 contiguous nucleotides that are identical to a sequence of 20 contiguous nucleotides in the unique sequence region of any one of the aptamer selected from the group of: SEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98 to 164, SEQ ID NO 165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS 212-214, ARC1115 (SEQ ID NO 221), ARC1172 (SEQ ID NO 222), ARC1194
  • the aptamer of the invention upon binding a von Willebrand Factor target modulates a von Willebrand Factor function, preferably in vivo, and comprises 8 contiguous nucleotides that are identical to a sequence of 8 contiguous nucleotides in the unique sequence region of any one of the aptamer selected from the group of: SEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98 to 164, SEQ ID NO 165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS 212-214, ARC1115 (SEQ ID NO 221), ARC1172 (SEQ ID NO 222) (SEQ ID NOS 212-214
  • the aptamer of the invention upon binding a von Willebrand Factor target modulates a von Willebrand Factor function, preferably in vivo, and comprises 4 contiguous nucleotides that are identical to a sequence of 4 contiguous nucleotides in the unique sequence region of any one of the aptamer selected from the group of: SEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98 to 164, SEQ ID NO 165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS 212-214, ARC1115 (SEQ ID NO 221), ARC1172 (SEQ ID NO 222) (SEQ ID NOS 212-214
  • a suitable method for generating an aptamer is with the process entitled “Systematic Evolution of Ligands by Exponential Enrichment” (“SELEXTM”) generally depicted in FIG. 1 .
  • SELEXTM Systematic Evolution of Ligands by Exponential Enrichment
  • the SELEXTM process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in, e.g., U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, now abandoned, U.S. Pat. No. 5,475,096 entitled “Nucleic Acid Ligands”, and U.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled “Nucleic Acid Ligands”.
  • Each SELEXTM-identified nucleic acid ligand i.e., each aptamer, is a specific ligand of a given target compound or molecule.
  • the SELEXTM process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (i.e., form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets.
  • SELEXTM relies as a starting point upon a large library or pool of single stranded oligonucleotides comprising randomized sequences.
  • the oligonucleotides can be modified or unmodified DNA, RNA, or DNA/RNA hybrids.
  • the pool comprises 100% random or partially random oligonucleotides.
  • the pool comprises random or partially random oligonucleotides containing at least one fixed sequence and/or conserved sequence incorporated within randomized sequence.
  • the pool comprises random or partially random oligonucleotides containing at least one fixed and/or conserved sequence at its 5′ and/or 3′ end which may comprise a sequence shared by all the molecules of the oligonucleotide pool.
  • Fixed sequences are sequences common to oligonucleotides in the pool which are incorporated for a preselected purpose, such as CpG motifs described further below, hybridization sites for PCR primers, promoter sequences for RNA polymerases (e.g., T3, T4, T7, and SP6), restriction sites, or homopolymeric sequences, such as poly A or poly T tracts, catalytic cores, sites for selective binding to affinity columns, and other sequences to facilitate cloning and/or sequencing of an oligonucleotide of interest.
  • conserveed sequences are sequences, other than the previously described fixed sequences, shared by a number of aptamers that bind to the same target.
  • the oligonucleotides of the pool preferably include a randomized sequence portion as well as fixed sequences necessary for efficient amplification.
  • the oligonucleotides of the starting pool contain fixed 5′ and 3′ terminal sequences which flank an internal region of 30-50 random nucleotides.
  • the randomized nucleotides can be produced in a number of ways including chemical synthesis and size selection from randomly cleaved cellular nucleic acids. Sequence variation in test nucleic acids can also be introduced or increased by mutagenesis before or during the selection/amplification iterations.
  • the random sequence portion of the oligonucleotide can be of any length and can comprise ribonucleotides and/or deoxyribonucleotides and can include modified or non-natural nucleotides or nucleotide analogs. See, e.g., U.S. Pat. No. 5,958,691; U.S. Pat. No. 5,660,985; U.S. Pat. No. 5,958,691; U.S. Pat. No. 5,698,687; U.S. Pat. No. 5,817,635; U.S. Pat. No. 5,672,695, and PCT Publication WO 92/07065.
  • Random oligonucleotides can be synthesized from phosphodiester-linked nucleotides using solid phase oligonucleotide synthesis techniques well known in the art. See, e.g., Froehler et al., Nucl. Acid Res. 14:5399-5467 (1986) and Froehler et al., Tet. Lett. 27:5575-5578 (1986). Random oligonucleotides can also be synthesized using solution phase methods such as triester synthesis methods. See, e.g., Sood et al., Nucl. Acid Res. 4:2557 (1977) and Hirose et al., Tet. Lett., 28:2449 (1978).
  • the starting library of oligonucleotides may be generated by automated chemical synthesis on a DNA synthesizer. To synthesize randomized sequences, mixtures of all four nucleotides are added at each nucleotide addition step during the synthesis process, allowing for random incorporation of nucleotides. As stated above, in one embodiment, random oligonucleotides comprise entirely random sequences; however, in other embodiments, random oligonucleotides can comprise stretches of nonrandom or partially random sequences. Partially random sequences can be created by adding the four nucleotides in different molar ratios at each addition step.
  • the starting library of oligonucleotides may be either RNA or DNA, or substituted RNA or DNA.
  • an RNA library is to be used as the starting library it is typically generated by synthesizing a DNA library, optionally PCR amplifying, then transcribing the DNA library in vitro using T7 RNA polymerase or modified T7 RNA polymerases, and purifying the transcribed library.
  • the RNA or DNA library is then mixed with the target under conditions favorable for binding and subjected to step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity.
  • the SELEXTM method includes steps of: (a) contacting the mixture with the target under conditions favorable for binding; (b) partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules; (c) dissociating the nucleic acid-target complexes; (d) amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids; and (e) reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the target molecule.
  • the SELEXTM method further comprises the steps of: (i) reverse transcribing the nucleic acids dissociated from the nucleic acid-target complexes before amplification in step (d); and (ii) transcribing the amplified nucleic acids from step (d) before restarting the process.
  • a nucleic acid mixture comprising, for example, a 20 nucleotide randomized segment can have 4 20 candidate possibilities. Those which have the higher affinity (lower dissociation constants) for the target are most likely to bind to the target.
  • a second nucleic acid mixture is generated, enriched for the higher binding affinity candidates. Additional rounds of selection progressively favor the best ligands until the resulting nucleic acid mixture is predominantly composed of only one or a few sequences. These can then be cloned, sequenced and individually tested for binding affinity as pure ligands or aptamers.
  • Cycles of selection and amplification are repeated until a desired goal is achieved. In the most general case, selection/amplification is continued until no significant improvement in binding strength is achieved on repetition of the cycle.
  • the method is typically used to sample approximately 10 14 different nucleic acid species but may be used to sample as many as about 10 18 different nucleic acid species.
  • nucleic acid aptamer molecules are selected in a 5 to 20 cycle procedure. In one embodiment, heterogeneity is introduced only in the initial selection stages and does not occur throughout the replicating process.
  • the selection process is so efficient at isolating those nucleic acid ligands that bind most strongly to the selected target, that only one cycle of selection and amplification is required.
  • Such an efficient selection may occur, for example, in a chromatographic-type process wherein the ability of nucleic acids to associate with targets bound on a column operates in such a manner that the column is sufficiently able to allow separation and isolation of the highest affinity nucleic acid ligands.
  • the target-specific nucleic acid ligand solution may include a family of nucleic acid structures or motifs that have a number of conserved sequences and a number of sequences which can be substituted or added without significantly affecting the affinity of the nucleic acid ligands to the target.
  • nucleic acid primary, secondary and tertiary structures are known to exist.
  • the structures or motifs that have been shown most commonly to be involved in non-Watson-Crick type interactions are referred to as hairpin loops, symmetric and asymmetric bulges, pseudoknots and myriad combinations of the same.
  • Almost all known cases of such motifs suggest that they can be formed in a nucleic acid sequence of no more than 30 nucleotides. For this reason, it is often preferred that SELEXTM procedures with contiguous randomized segments be initiated with nucleic acid sequences containing a randomized segment of between about 20 to about 50 nucleotides, and of about 30 to about 40 nucleotides in some embodiments.
  • the 5′-fixed:random:3′-fixed sequence comprises a random sequence of about 30 to about 50 nucleotides.
  • U.S. Pat. No. 5,707,796 describes the use of SELEXTM in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA.
  • U.S. Pat. No. 5,763,177 describes SELEXTM based methods for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule.
  • SELEXTM can also be used to obtain nucleic acid ligands that bind to more than one site on the target molecule, and to obtain nucleic acid ligands that include non-nucleic acid species that bind to specific sites on the target.
  • SELEXTM provides means for isolating and identifying nucleic acid ligands which bind to any envisionable target, including large and small biomolecules such as nucleic acid-binding proteins and proteins not known to bind nucleic acids as part of their biological function as well as cofactors and other small molecules.
  • U.S. Pat. No. 5,580,737 discloses nucleic acid sequences identified through SELEXTM which are capable of binding with high affinity to caffeine and the closely related analog, theophylline.
  • Counter-SELEXTM is a method for improving the specificity of nucleic acid ligands to a target molecule by eliminating nucleic acid ligand sequences with cross-reactivity to one or more non-target molecules.
  • Counter-SELEXTM is comprised of the steps of: (a) preparing a candidate mixture of nucleic acids; (b) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; (c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; (d) dissociating the increased affinity nucleic acids from the target; (e) contacting the increased affinity nucleic acids with one or more non-target molecules such that nucleic acid ligands with specific affinity for the non-target molecule(s) are removed; and (f) amplifying the nucleic acids with specific affinity only to the target molecule to yield a mixture of nucleic acids enriched for nucleic acid sequences with
  • oligonucleotides in their phosphodiester form may be quickly degraded in body fluids by intracellular and extracellular enzymes such as endonucleases and exonucleases before the desired effect is manifest.
  • the SELEXTM method thus encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the sugar and/or phosphate and/or base positions.
  • SELEXTM-identified nucleic acid ligands containing modified nucleotides are described, e.g., in U.S.
  • Pat. No. 5,660,985 which describes oligonucleotides containing nucleotide derivatives chemically modified at the 2′ position of ribose, 5 position of pyrimidines, and 8 position of purines
  • U.S. Pat. No. 5,756,703 which describes oligonucleotides containing various 2′-modified pyrimidines
  • U.S. Pat. No. 5,580,737 which describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2′-amino (2′-NH 2 ), 2′-fluoro (2′-F), and/or 2′-OMe substituents.
  • Modifications of the nucleic acid ligands contemplated in this invention include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Modifications to generate oligonucleotide populations which are resistant to nucleases can also include one or more substitute internucleotide linkages, altered sugars, altered bases, or combinations thereof.
  • modifications include, but are not limited to, 2′-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbone modifications, phosphorothioate or alkyl phosphate modifications, methylations, and unusual base-pairing combinations such as the isobases isocytidine and isoguanidine. Modifications can also include 3′ and 5′ modifications such as capping.
  • oligonucleotides are provided in which the P(O)O group is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), P(O)NR 2 (“amidate”), P(O)R, P(O)OR′, CO or CH 2 (“formacetal”) or 3′-amine (—NH—CH 2 —CH 2 —), wherein each R or R′ is independently H or substituted or unsubstituted alkyl.
  • Linkage groups can be attached to adjacent nucleotides through an —O-, —N-, or —S-linkage. Not all linkages in the oligonucleotide are required to be identical.
  • the term phosphorothioate encompasses one or more non-bridging oxygen atoms in a phosphodiester bond replaced by one or more sulfur atoms
  • the oligonucleotides comprise modified sugar groups, for example, one or more of the hydroxyl groups is replaced with halogen, aliphatic groups, or functionalized as ethers or amines.
  • the 2′-position of the furanose residue is substituted by any of an O-methyl, O-alkyl, O-allyl, S-alkyl, S-allyl, or halo group.
  • modifications are known to one of ordinary skill in the art. Such modifications may be pre-SELEXTM process modifications or post-SELEXTM process modifications (modification of previously identified unmodified ligands) or may be made by incorporation into the SELEXTM process.
  • Pre-SELEXTM process modifications or those made by incorporation into the SELEXTM process yield nucleic acid ligands with both specificity for their SELEXTM target and improved stability, e.g., in vivo stability.
  • Post-SELEXTM process modifications made to nucleic acid ligands may result in improved stability, e.g., in vivo stability without adversely affecting the binding capacity of the nucleic acid ligand.
  • the SELEXTM method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. Pat. No. 5,637,459 and U.S. Pat. No. 5,683,867.
  • the SELEXTM method further encompasses combining selected nucleic acid ligands with lipophilic or non-immunogenic high molecular weight compounds in a diagnostic or therapeutic complex, as described, e.g., in U.S. Pat. No. 6,011,020, U.S. Pat. No. 6,051,698, and PCT Publication No. WO 98/18480.
  • These patents and applications teach the combination of a broad array of shapes and other properties, with the efficient amplification and replication properties of oligonucleotides, and with the desirable properties of other molecules.
  • nucleic acid ligands to small, flexible peptides via the SELEXTM method has also been explored.
  • Small peptides have flexible structures and usually exist in solution in an equilibrium of multiple conformers, and thus it was initially thought that binding affinities may be limited by the conformational entropy lost upon binding a flexible peptide.
  • binding affinities may be limited by the conformational entropy lost upon binding a flexible peptide.
  • the feasibility of identifying nucleic acid ligands to small peptides in solution was demonstrated in U.S. Pat. No. 5,648,214 in which high affinity RNA nucleic acid ligands to substance P, an 11 amino acid peptide, were identified.
  • the aptamers with specificity and binding affinity to the target(s) of the present invention are typically selected by the SELEXTM process as described herein. As part of the SELEXTM process, the sequences selected to bind to the target are then optionally minimized to determine the minimal sequence having the desired binding affinity.
  • the selected aptamer sequences and/or the minimized aptamer sequences are optionally optimized by performing random or directed mutagenesis of the sequence to increase binding affinity or alternatively to determine which positions in the sequence are essential for binding activity. For example, a “doped reselections” may be used to explore the sequence requirements within an aptamer.
  • selections are carried out with a synthetic, degenerate pool that has been designed based on a single sequence.
  • the level of degeneracy usually varies from 70% to 85% wild type nucleotide.
  • neutral mutations are observed following doped reselection but in some cases sequence changes can result in improvements in affinity.
  • selections can be performed with sequences incorporating modified sequences to stabilize the aptamer molecules against degradation in vivo.
  • an aptamer In order for an aptamer to be suitable for use as a therapeutic, it is preferably inexpensive to synthesize, safe and stable in vivo. Wild-type RNA and DNA aptamers are typically not stable in vivo because of their susceptibility to degradation by nucleases. Resistance to nuclease degradation can be greatly increased, if desired, by the incorporation of modifying groups at the 2′-position.
  • Aptamers that contain 2′-O-methyl (“2′-OMe”) nucleotides overcome many of these drawbacks.
  • Oligonucleotides containing 2′-OMe nucleotides are nuclease-resistant and inexpensive to synthesize.
  • 2′-OMe nucleotides are ubiquitous in biological systems, natural polymerases do not accept 2′-OMe NTPs as substrates under physiological conditions, thus there are no safety concerns over the recycling of 2′-OMe nucleotides into host DNA.
  • the SELEXTM methods used to generate 2′-modified aptamers is described, e.g., in U.S. Provisional Patent Application Ser. No. 60/430,761, filed Dec.
  • the present invention includes aptamers that bind to and modulate the function of von Willebrand Factor which contain modified nucleotides (e.g., nucleotides which have a modification at the 2′position) to make the oligonucleotide more stable than the unmodified oligonucleotide to enzymatic and chemical degradation as well as thermal and physical degradation.
  • modified nucleotides e.g., nucleotides which have a modification at the 2′position
  • aptamers generated using this method tend to contain from two to four 2′-OH residues, and stability and cost of synthesis are compromised as a result.
  • modified nucleotides into the transcription reaction which generate stabilized oligonucleotides used in oligonucleotide libraries from which aptamers are selected and enriched by SELEXTM (and/or any of its variations and improvements, including those described herein)
  • the methods of the present invention eliminate the need for stabilizing the selected aptamer oligonucleotides (e.g., by resynthesizing the aptamer oligonucleotides with modified nucleotides).
  • the present invention provides aptamers comprising combinations of 2′-OH, 2′-F, 2′-deoxy, and 2′-OMe modifications of the ATP, GTP, CTP, TTP, and UTP nucleotides.
  • the present invention provides aptamers comprising combinations of 2′-OH, 2′-F, 2′-deoxy, 2′-OMe, 2′-NH 2 , and 2′-methoxyethyl modifications of the ATP, GTP, CTP, TTP, and UTP nucleotides.
  • the present invention provides aptamers comprising 5 6 combinations of 2′-OH, 2′-F, 2′-deoxy, 2′-OMe, 2′-NH 2 , and 2′-methoxyethyl modifications of the ATP, GTP, CTP, TTP, and UTP nucleotides.
  • 2′-modified aptamers of some embodiments of the invention are created using modified polymerases, e.g., a modified T7 polymerase, having a rate of incorporation of modified nucleotides having bulky substituents at the furanose 2′ position that is higher than that of wild-type polymerases.
  • modified polymerases e.g., a modified T7 polymerase
  • Y639F single mutant T7 polymerase
  • NTPs 2′deoxy, 2′amino-, and 2′fluoro-nucleotide triphosphates
  • this mutant T7 polymerase reportedly can not readily utilize (i.e., incorporate) NTPs with bulky 2′-substituents such as 2′-OMe or 2′-azido (2′-N 3 ) substituents.
  • bulky 2′ substituents such as 2′-OMe or 2′-azido (2′-N 3 ) substituents.
  • a double T7 polymerase mutant (Y639F/H784A) having the histidine at position 784 changed to an alanine residue in addition to the Y639F mutation has been described and has been used in limited circumstances to incorporate modified pyrimidine NTPs. See Padilla, R. and Sousa, R., Nucleic Acids Res., 2002, 30(24): 138.
  • a Y639F/H784A/K378R mutant T7 RNA polymerase has been used in limited circumstances to incorporate modified purine and pyrimidine NTPs, e.g., 2′-OMe NPTs, but requires a spike of 2′-OH GTP for transcription. See Burmeister et. al., Chemistry and Biology, 2005, 12: 25-33.
  • a single mutant T7 polymerase (H784A) having the histidine at position 784 changed to an alanine residue has also been described. Padilla et al., Nucleic Acids Research, 2002, 30: 138.
  • the Y693F single mutant can be used for the incorporation of all 2′-OMe substituted NTPs except GTP and the Y639F/H784A, Y639F/H784A/K378R, Y639L/H784A, and Y639L/H784A/K378R mutant T7 RNA polymerases can be used for the incorporation of all 2′-OMe substituted NTPs including GTP. It is expected that the H784A single mutant possesses properties similar to the Y639F and the Y639F/H784A mutants when used under the conditions disclosed herein.
  • 2′-modified oligonucleotides may be synthesized entirely of modified nucleotides, or with a subset of modified nucleotides. All nucleotides may be modified, and all may contain the same modification. All nucleotides may be modified, but contain different modifications, e.g., all nucleotides containing the same base may have one type of modification, while nucleotides containing other bases may have different types of modification. All purine nucleotides may have one type of modification (or are unmodified), while all pyrimidine nucleotides have another, different type of modification (or are unmodified).
  • transcripts, or libraries of transcripts are generated using any combination of modifications, including for example, ribonucleotides (2′-OH), deoxyribonucleotides (2′-deoxy), 2′-F, and 2′-OMe nucleotides.
  • a transcription mixture containing 2′-OMe C and U and 2′-OH A and G is referred to as a “rRmY” mixture and aptamers selected therefrom are referred to as “rRmY” aptamers.
  • a transcription mixture containing deoxy A and G and 2′-OMe U and C is referred to as a “dRmY” mixture and aptamers selected therefrom are referred to as “dRmY” aptamers.
  • a transcription mixture containing 2′-OMe A, C, and U, and 2′-OH G is referred to as a “rGmH” mixture and aptamers selected therefrom are referred to as “rGmH” aptamers.
  • a transcription mixture alternately containing 2′-OMe A, C, U and G and 2′-OMe A, U and C and 2′-F G is referred to as a “alternating mixture” and aptamers selected therefrom are referred to as “alternating mixture” aptamers.
  • a transcription mixture containing 2′-OMe A, U, C, and G, where up to 10% of the G's are ribonucleotides is referred to as a “r/mGmH” mixture and aptamers selected therefrom are referred to as “r/mGmH” aptamers.
  • a transcription mixture containing 2′-OMe A, U, and C, and 2′-F G is referred to as a “fGmH” mixture and aptamers selected therefrom are referred to as “fGmH” aptamers.
  • a transcription mixture containing 2′-OMe A, U, and C, and deoxy G is referred to as a “dGmH” mixture and aptamers selected therefrom are referred to as “dGmH” aptamers.
  • a transcription mixture containing deoxy A, and 2′-OMe C, G and U is referred to as a “dAmB” mixture and aptamers selected therefrom are referred to as “dAmB” aptamers
  • a transcription mixture containing all 2′-OH nucleotides is referred to as a “rN” mixture and aptamers selected therefrom are referred to as “rN”, “rRrY”, or “RNA” aptamers.
  • a transcription mixture containing 2′-OH adenosine triphosphate and guanosine triphosphate and deoxy cytidine triphosphate and thymidine triphosphate is referred to as a rRdY mixture and aptamers selected therefrom are referred to as “rRdY” aptamers.
  • rRdY aptamers selected therefrom are referred to as “rRdY” aptamers.
  • a “mRmY” aptamer is one containing only 2′-OMe nucleotides except for the starting nucleotide which is 2′-hydroxy.
  • a preferred embodiment includes any combination of 2′-OH, 2′-deoxy and 2′-OMe nucleotides. Another embodiment includes any combination of 2′-deoxy and 2′-OMe nucleotides. Yet another embodiment includes any combination of 2′-deoxy and 2′-OMe nucleotides in which the pyrimidines are 2′-OMe (such as dRmY, mRmY or dGmH).
  • Incorporation of modified nucleotides into the aptamers of the invention may be accomplished before (pre-) the selection process (e.g., a pre-SELEXTM process modification).
  • aptamers of the invention in which modified nucleotides have been incorporated by pre-SELEXTM process modification can be further modified by a post-SELEXTM modification process (i.e., a post-SELEXTM process modification after a pre-SELEXTM modification).
  • Pre-SELEXTM process modifications yield modified nucleic acid ligands with specificity for the SELEXTM target and also improved in vivo stability.
  • Post-SELEXTM process modifications i.e., modification (e.g., truncation, deletion, substitution or additional nucleotide modifications of previously identified ligands having nucleotides incorporated by pre-SELEXTM process modification) can result in a further improvement of in vivo stability without adversely affecting the binding capacity of the nucleic acid ligand having nucleotides incorporated by pre-SELEXTM process modification.
  • modification e.g., truncation, deletion, substitution or additional nucleotide modifications of previously identified ligands having nucleotides incorporated by pre-SELEXTM process modification
  • RNA transcripts in conditions under which a polymerase accepts 2′-modified NTPs the Y693F, Y693F/K378R, Y693F/H784A, Y693F/H784A/K378R, Y693L/H784A, Y693L/H784A/K378R Y639L, or the Y639L/K378Rmutant T7 RNA polymerases can be used.
  • a preferred polymerase is the Y639L/H784A mutant T7 RNA polymerase.
  • T7 RNA polymerase is the Y639L/H784A/K378R mutant T7 RNA polymerase.
  • Other T7 RNA polymerases particularly those that exhibit a high tolerance for bulky 2′-substituents, may also be used in the present invention.
  • the Y639L/H784A or the Y639L/H784A/K378R mutant T7 RNA polymerase can be used for the incorporation of all 2′-OMe NTPs, including GTP, with higher transcript yields than achieved by using the Y639F, Y639F/K378R, Y639F/H784A, Y639F/H784A/K378R, Y639L, or the Y639L/K378R mutant T7 RNA polymerases.
  • the Y639L/H784A and Y639L/H784A/K378R mutant T7 RNA polymerases can be used with but does not require 2′-OH GTP to achieve high yields of 2′-modified, e.g., 2′-OMe containing oligonucleotides.
  • leader sequence is typically 6-15 nucleotides long, and may be composed of all purines, or a mixture of purine and pyrimidine nucleotides.
  • Transcription can be divided into two phases: the first phase is initiation, during which an NTP is added to the 3′-hydroxyl end of GTP (or another substituted guanosine) to yield a dinucleotide which is then extended by about 10-12 nucleotides; the second phase is elongation, during which transcription proceeds beyond the addition of the first about 10-12 nucleotides.
  • concentrations of approximately 5 mM magnesium chloride and 1.5 mM manganese chloride are preferred when each NTP is present at a concentration of 0.5 mM.
  • concentrations of approximately 6.5 mM magnesium chloride and 2.0 mM manganese chloride are preferred.
  • concentrations of approximately 9.5 mM magnesium chloride and 3.0 mM manganese chloride are preferred. In any case, departures from these concentrations of up to two-fold still give significant amounts of modified transcripts.
  • one unit of the Y639F/H784A mutant T7 RNA polymerase is defined as the amount of enzyme required to incorporate 1 nmole of 2′-OMe NTPs into transcripts under the r/mGmH conditions.
  • one unit of inorganic pyrophosphatase is defined as the amount of enzyme that will liberate 1.0 mole of inorganic orthophosphate per minute at pH 7.2 and 25° C.
  • the leader sequence is an all purine leader sequence. In another embodiment, the leader sequence is a mixture of purines and pyrimidines. As used herein, one unit of inorganic pyrophosphatase is defined as the amount of enzyme that will liberate 1.0 mole of inorganic orthophosphate per minute at pH 7.2 and 25° C.
  • transcription is preferably performed at a temperature of from about 20° C. to about 50° C., preferably from about 30° C. to 45° C., and more preferably at about 37° C. for a period of at least two hours and (b) 50-300 nM of a double stranded DNA transcription template is used (200 nM template is used in round 1 to increase diversity (300 nM template is used in dRmY transcriptions)), and for subsequent rounds approximately 50 nM, a 1/10 dilution of an optimized PCR reaction, using conditions described herein, is used).
  • the preferred DNA transcription templates are described below (where ARC254 and ARC256 transcribe under all 2′-OMe conditions and ARC255 transcribes under rRmY conditions).
  • the transcription reaction mixture comprises 2′-OH adenosine triphosphates (ATP), 2′-OH guanosine triphosphates (GTP), 2′-OH cytidine triphosphates (CTP), and 2′-OH uridine triphosphates (UTP).
  • the modified oligonucleotides produced using the rN transcription mixtures of the present invention comprise substantially all 2′-OH adenosine, 2′-OH guanosine, 2′-OH cytidine, and 2′-OH uridine.
  • the resulting modified oligonucleotides comprise a sequence where at least 80% of all adenosine nucleotides are 2′-OH adenosine, at least 80% of all guanosine nucleotides are 2′-OH guanosine, at least 80% of all cytidine nucleotides are 2′-OH cytidine, and at least 80% of all uridine nucleotides are 2′-OH uridine.
  • the resulting modified oligonucleotides of the present invention comprise a sequence where at least 90% of all adenosine nucleotides are 2′-OH adenosine, at least 90% of all guanosine nucleotides are 2′-OH guanosine, at least 90% of all cytidine nucleotides are 2′-OH cytidine, and at least 90% of all uridine nucleotides are 2′-OH uridine.
  • the modified oligonucleotides of the present invention comprise a sequence where 100% of all adenosine nucleotides are 2′-OH adenosine, 100% of all guanosine nucleotides are 2′-OH guanosine, 100% of all cytidine nucleotides are 2′-OH cytidine, and 100% of all uridine nucleotides are 2′-OH uridine.
  • the transcription reaction mixture comprises 2′-OH adenosine triphosphates, 2′-OH guanosine triphosphates, 2′-OMe cytidine triphosphates, and 2′-OMe uridine triphosphates.
  • the modified oligonucleotides produced using the rRmY transcription mixtures of the present invention comprise substantially all 2′-OH adenosine, 2′-OH guanosine, 2′-OMe cytidine and 2′-OMe uridine.
  • the resulting modified oligonucleotides comprise a sequence where at least 80% of all adenosine nucleotides are 2′-OH adenosine, at least 80% of all guanosine nucleotides are 2′-OH guanosine, at least 80% of all cytidine nucleotides are 2′-OMe cytidine and at least 80% of all uridine nucleotides are 2′-OMe uridine.
  • the resulting modified oligonucleotides comprise a sequence where at least 90% of all adenosine nucleotides are 2′-OH adenosine, at least 90% of all guanosine nucleotides are 2′-OH guanosine, at least 90% of all cytidine nucleotides are 2′-OMe cytidine and at least 90% of all uridine nucleotides are 2′-OMe uridine
  • the resulting modified oligonucleotides comprise a sequence where 100% of all adenosine nucleotides are 2′-OH adenosine, 100% of all guanosine nucleotides are 2′-OH guanosine, 100% of all cytidine nucleotides are 2′-OMe cytidine and 100% of all uridine nucleotides are 2′-OMe uridine.
  • the transcription reaction mixture comprises 2′-deoxy adenosine triphosphates, 2′-deoxy guanosine triphosphates, 2′-O-methyl cytidine triphosphates, and 2′-O-methyl uridine triphosphates.
  • the modified oligonucleotides produced using the dRmY transcription conditions of the present invention comprise substantially all 2′-deoxy adenosine, 2′-deoxy guanosine, 2′-O-methyl cytidine, and 2′-O-methyl uridine.
  • the resulting modified oligonucleotides of the present invention comprise a sequence where at least 80% of all adenosine nucleotides are 2′-deoxy adenosine, at least 80% of all guanosine nucleotides are 2′-deoxy guanosine, at least 80% of all cytidine nucleotides are 2′-O-methyl cytidine, and at least 80% of all uridine nucleotides are 2′-O-methyl uridine.
  • the resulting modified oligonucleotides of the present invention comprise a sequence where at least 90% of all adenosine nucleotides are 2′-deoxy adenosine, at least 90% of all guanosine nucleotides are 2′-deoxy guanosine, at least 90% of all cytidine nucleotides are 2′-O-methyl cytidine, and at least 90% of all uridine nucleotides are 2′-O-methyl uridine.
  • the resulting modified oligonucleotides of the present invention comprise a sequence where 100% of all adenosine nucleotides are 2′-deoxy adenosine, 100% of all guanosine nucleotides are 2′-deoxy guanosine, 100% of all cytidine nucleotides are 2′-O-methyl cytidine, and 100% of all uridine nucleotides are 2′-O-methyl uridine.
  • the transcription reaction mixture comprises 2′-OH guanosine triphosphates, 2′-O-methyl cytidine triphosphates, 2′-O-methyl uridine triphosphates, and 2′-O-methyl adenosine triphosphates.
  • the modified oligonucleotides produced using the rGmH transcription mixtures of the present invention comprise substantially all 2′-OH guanosine, 2′-O-methyl cytidine, 2′-O-methyl uridine, and 2′-O-methyl adenosine.
  • the resulting modified oligonucleotides comprise a sequence where at least 80% of all guanosine nucleotides are 2′-OH guanosine, at least 80% of all cytidine nucleotides are 2′-O-methyl cytidine, at least 80% of all uridine nucleotides are 2′-O-methyl uridine, and at least 80% of all adenosine nucleotides are 2′-O-methyl adenosine.
  • the resulting modified oligonucleotides comprise a sequence where at least 90% of all guanosine nucleotides are 2′-OH guanosine, at least 90% of all cytidine nucleotides are 2′-O-methyl cytidine, at least 90% of all uridine nucleotides are 2′-O-methyl uridine, and at least 90% of all adenosine nucleotides are 2′-O-methyl adenosine.
  • the resulting modified oligonucleotides comprise a sequence where 100% of all guanosine nucleotides are 2′-OH guanosine, 100% of all cytidine nucleotides are 2′-O-methyl cytidine, 100% of all uridine nucleotides are 2′-O-methyl uridine, and 100% of all adenosine nucleotides are 2′-O-methyl adenosine.
  • the transcription reaction mixture comprises 2′-O-methyl adenosine triphosphate, 2′-O-methyl cytidine triphosphate, 2′-O-methyl guanosine triphosphate, 2′-O-methyl uridine triphosphate and 2′-OH guanosine triphosphate.
  • the resulting modified oligonucleotides produced using the r/mGmH transcription mixtures of the present invention comprise substantially all 2′-O-methyl adenosine, 2′-O-methyl cytidine, 2′-O-methyl guanosine, and 2′-O-methyl uridine, wherein the population of guanosine nucleotides has a maximum of about 10% 2′-OH guanosine.
  • the resulting r/mGmH modified oligonucleotides of the present invention comprise a sequence where at least 80% of all adenosine nucleotides are 2′-O-methyl adenosine, at least 80% of all cytidine nucleotides are 2′-O-methyl cytidine, at least 80% of all guanosine nucleotides are 2′-O-methyl guanosine, at least 80% of all uridine nucleotides are 2′-O-methyl uridine, and no more than about 10% of all guanosine nucleotides are 2′-OH guanosine.
  • the resulting modified oligonucleotides comprise a sequence where at least 90% of all adenosine nucleotides are 2′-O-methyl adenosine, at least 90% of all cytidine nucleotides are 2′-O-methyl cytidine, at least 90% of all guanosine nucleotides are 2′-O-methyl guanosine, at least 90% of all uridine nucleotides are 2′-O-methyl uridine, and no more than about 10% of all guanosine nucleotides are 2′-OH guanosine.
  • the resulting modified oligonucleotides comprise a sequence where 100% of all adenosine nucleotides are 2′-O-methyl adenosine, 100% of all cytidine nucleotides are 2′-O-methyl cytidine, 90% of all guanosine nucleotides are 2′-O-methyl guanosine, and 100% of all uridine nucleotides are 2′-O-methyl uridine, and no more than about 10% of all guanosine nucleotides are 2′-OH guanosine.
  • the transcription mixture comprises only 2′-O-methyl adenosine triphosphate, 2′-O-methyl cytidine triphosphate, 2′-O-methyl guanosine triphosphate, 2′-O-methyl uridine triphosphate.
  • the resulting modified oligonucleotides produced using the mRmY transcription mixture of the present invention comprise a sequence where 100% of all adenosine nucleotides are 2′-O-methyl adenosine, 100% of all cytidine nucleotides are 2′-O-methyl cytidine, 100% of all guanosine nucleotides are 2′-O-methyl guanosine, and 100% of all uridine nucleotides are 2′-O-methyl uridine.
  • the transcription reaction mixture comprises 2′-O-methyl adenosine triphosphates, 2′-O-methyl uridine triphosphates, 2′-O-methyl cytidine triphosphates, and 2′-F guanosine triphosphates.
  • the modified oligonucleotides produced using the fGmH transcription conditions of the present invention comprise substantially all 2′-O-methyl adenosine, 2′-O-methyl uridine, 2′-O-methyl cytidine, and 2′-F guanosine.
  • the resulting modified oligonucleotides comprise a sequence where at least 80% of all adenosine nucleotides are 2′-O-methyl adenosine, at least 80% of all uridine nucleotides are 2′-O-methyl uridine, at least 80% of all cytidine nucleotides are 2′-O-methyl cytidine, and at least 80% of all guanosine nucleotides are 2′-F guanosine.
  • the resulting modified oligonucleotides comprise a sequence where at least 90% of all adenosine nucleotides are 2′-O-methyl adenosine, at least 90% of all uridine nucleotides are 2′-O-methyl uridine, at least 90% of all cytidine nucleotides are 2′-O-methyl cytidine, and at least 90% of all guanosine nucleotides are 2′-F guanosine.
  • the resulting modified oligonucleotides comprise a sequence where 100% of all adenosine nucleotides are 2′-O-methyl adenosine, 100% of all uridine nucleotides are 2′-O-methyl uridine, 100% of all cytidine nucleotides are 2′-O-methyl cytidine, and 100% of all guanosine nucleotides are 2′-F guanosine.
  • the transcription reaction mixture comprises 2′-deoxy adenosine triphosphates, 2′-O-methyl cytidine triphosphates, 2′-O-methyl guanosine triphosphates, and 2′-O-methyl uridine triphosphates.
  • the modified oligonucleotides produced using the dAmB transcription mixtures of the present invention comprise substantially all 2′-deoxy adenosine, 2′-O-methyl cytidine, 2′-O-methyl guanosine, and 2′-O-methyl uridine.
  • the resulting modified oligonucleotides comprise a sequence where at least 80% of all adenosine nucleotides are 2′-deoxy adenosine, at least 80% of all cytidine nucleotides are 2′-O-methyl cytidine, at least 80% of all guanosine nucleotides are 2′-O-methyl guanosine, and at least 80% of all uridine nucleotides are 2′-O-methyl uridine.
  • the resulting modified oligonucleotides comprise a sequence where at least 90% of all adenosine nucleotides are 2′-deoxy adenosine, at least 90% of all cytidine nucleotides are 2′-O-methyl cytidine, at least 90% of all guanosine nucleotides are 2′-O-methyl guanosine, and at least 90% of all uridine nucleotides are 2′-O-methyl uridine.
  • the resulting modified oligonucleotides of the present invention comprise a sequence where 100% of all adenosine nucleotides are 2′-deoxy adenosine, 100% of all cytidine nucleotides are 2′-O-methyl cytidine, 100% of all guanosine nucleotides are 2′-O-methyl guanosine, and 100% of all uridine nucleotides are 2′-O-methyl uridine.
  • the transcription products can then be used for input into the SELEXTM process to identify aptamers and/or to determine a conserved sequences that has binding specificity to a given target.
  • the resulting sequences are already partially stabilized, eliminating this step from the post-SELEXTM process to arrive at an optimized aptamer sequence and giving a more highly stabilized aptamer as a result.
  • Another advantage of the 2′-OMe SELEXTM process is that the resulting sequences are likely to have fewer 2′-OH nucleotides required in the sequence, possibly none. To the extent 2′OH nucleotides remain they may be removed by performing post-SELEXTM modifications.
  • transcripts fully incorporating 2′ substituted nucleotides can be obtained under conditions other than the optimized conditions described above.
  • variations to the above transcription conditions include:
  • the HEPES buffer concentration can range from 0 to 1 M.
  • the present invention also contemplates the use of other buffering agents having a pKa between 5 and 10 including, for example, Tris-hydroxymethyl-aminomethane.
  • the DTT concentration can range from 0 to 400 mM.
  • the methods of the present invention also provide for the use of other reducing agents including, for example, mercaptoethanol.
  • the spermidine and/or spermine concentration can range from 0 to 20 mM.
  • the PEG-8000 concentration can range from 0 to 50% (w/v).
  • the methods of the present invention also provide for the use of other hydrophilic polymer including, for example, other molecular weight PEG or other polyalkylene glycols.
  • the Triton X-100 concentration can range from 0 to 0.1% (w/v).
  • the methods of the present invention also provide for the use of other non-ionic detergents including, for example, other detergents, including other Triton-X detergents.
  • the MgCl 2 concentration can range from 0.5 mM to 50 mM.
  • the MnCl 2 concentration can range from 0.15 mM to 15 mM.
  • Both MgCl 2 and MnCl 2 must be present within the ranges described and in a preferred embodiment are present in about a 10 to about 3 ratio of MgCl 2 :MnCl 2 , preferably, the ratio is about 3-5:1, more preferably, the ratio is about 3-4:1.
  • the 2′-OMe NTP concentration (each NTP) can range from 5 ⁇ M to 5 mM.
  • the 2′-OH GTP concentration can range from 0 ⁇ M to 300 ⁇ M.
  • the 2′-OH GMP concentration can range from 0 to 5 mM.
  • the pH can range from pH 6 to pH 9.
  • the methods of the present invention can be practiced within the pH range of activity of most polymerases that incorporate modified nucleotides.
  • the methods of the present invention provide for the optional use of chelating agents in the transcription reaction condition including, for example, EDTA, EGTA, and DTT.
  • Aptamer Medicinal Chemistry is an aptamer improvement technique in which sets of variant aptamers are chemically synthesized. These sets of variants typically differ from the parent aptamer by the introduction of a single substituent, and differ from each other by the location of this substituent. These variants are then compared to each other and to the parent. Improvements in characteristics may be profound enough that the inclusion of a single substituent may be all that is necessary to achieve a particular therapeutic criterion.
  • the information gleaned from the set of single variants may be used to design further sets of variants in which more than one substituent is introduced simultaneously.
  • all of the single substituent variants are ranked, the top 4 are chosen and all possible double (6), triple (4) and quadruple (1) combinations of these 4 single substituent variants are synthesized and assayed.
  • the best single substituent variant is considered to be the new parent and all possible double substituent variants that include this highest-ranked single substituent variant are synthesized and assayed.
  • Other strategies may be used, and these strategies may be applied repeatedly such that the number of substituents is gradually increased while continuing to identify further-improved variants.
  • Aptamer Medicinal Chemistry may be used particularly as a method to explore the local, rather than the global, introduction of substituents. Because aptamers are discovered within libraries that are generated by transcription, any substituents that are introduced during the SELEXTM process must be introduced globally. For example, if it is desired to introduce phosphorothioate linkages between nucleotides then they can only be introduced at every A (or every G, C, T, U etc.) (globally substituted). Aptamers which require phosphorothioates at some As (or some G, C, T, U etc.) (locally substituted) but cannot tolerate it at other As cannot be readily discovered by this process.
  • Aptamer Medicinal Chemistry schemes may include substituents that introduce steric bulk, hydrophobicity, hydrophilicity, lipophilicity, lipophobicity, positive charge, negative charge, neutral charge, zwitterions, polarizability, nuclease-resistance, conformational rigidity, conformational flexibility, protein-binding characteristics, mass etc.
  • Aptamer Medicinal Chemistry schemes may include base-modifications, sugar-modifications or phosphodiester linkage-modifications.
  • the vWF aptamers of the invention include aptamers developed through aptamer medicinal chemistry as described herein.
  • the materials of the present invention comprise a series of nucleic acid aptamers of 29 to 76 nucleotides in length which bind specifically to von Willebrand Factor.
  • materials of the present invention comprise a series of nucleic acid aptamers of 29 to 76 nucleotides in length which bind specifically to von Willebrand Factor and which functionally modulate, e.g., block, an activity of von Willebrand Factor in vivo and/or cell-based assays.
  • aptamers specifically capable of binding and modulating full length von Willebrand Factor and/or von Willebrand Factor domain A1 are set forth herein. These aptamers provide a low-toxicity, safe, and effective modality of treating and/or preventing cardiovascular diseases or disorders.
  • the aptamers of the invention are used in a method to treat and/or prevent coronary artery diseases, including any one of the disorders selected from the group consisting of: arterial thrombosis and acute coronary syndromes such as unstable angina and myocardial infarction which are known to be caused by or otherwise associated with von Willebrand Factor mediated platelet aggregation.
  • the aptamers of the invention are used in a method to treat and/or prevent coronary artery diseases, including any one of the disorders selected from the group consisting of: arterial thrombosis and acute coronary syndromes such as unstable angina and myocardial infarction which are known to be caused by or otherwise associated with von Willebrand Factor mediated platelet aggregation while minimizing bleeding side effects.
  • the aptamers of the invention are used in a method to treat and/or prevent peripheral vascular diseases which are known to be caused by or otherwise associated with von Willebrand Factor mediated platelet aggregation.
  • the aptamers of the invention are used in a method to treat and/or prevent peripheral vascular diseases which are known to be caused by or otherwise associated with von Willebrand Factor mediated platelet aggregation, preferably, while minimizing bleeding side effects.
  • the aptamers of the invention are used to treat and/or prevent cerebrovascular diseases, including any one of the disorders selected from the group consisting of: transient cerebral ischemic attack, stroke and carotid stenosis which are known to be caused by or otherwise associated with von Willebrand Factor mediated platelet aggregation, preferably, while minimizing bleeding side effects.
  • aptamers of the invention are useful to inhibit von Willebrand Factor mediated platelet aggregation in a subject prior to, during, and/or after a subject has undergone percutaneous coronary intervention including angioplasty, thrombolytic treatment or coronary bypass surgery.
  • Aptamers of the invention are also useful for maintaining blood vessel patency in a subject prior to, during and/or after the subject has undergone coronary bypass surgery.
  • the aptamers of the invention are also useful for treating a patient undergoing dialysis.
  • the aptamers of the invention are also useful for inhibiting von Willebrand Factor mediated thrombosis in a subject, preferably while also minimizing bleeding side effects.
  • the thrombosis to be treated and/or inhibited may be associated with an inflammatory response.
  • the von Willebrand Factor specific binding aptamer for use in therapeutics and/or diagnostics is selected from the group consisting of: SEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98 to 164, SEQ ID NO 165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS 212-214, ARC11115 (SEQ ID NO 221), ARC172 (SEQ ID NO 222), ARC1194 (SEQ ID NO 223) to ARC1240 (SEQ ID NO 269), ARC1338 (SEQ ID NO 273) to ARC1346 (SEQ ID NO 281), ARC1361 (SEQ ID NO 284) to
  • von Willebrand Factor specific binding aptamers for use as therapeutics and/or diagnostics include any one of the following sequences: SEQ ID NO 23, SEQ ID NO 44, SEQ ID NO 49, SEQ ID NOS 98-100, SEQ ID NO 106, SEQ ID NO 109, SEQ ID NOS 114 to 115, SEQ ID NO 118, SEQ ID NO 127, SEQ ID NO 134, SEQ ID NO 164, SEQ ID NO165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 208, and SEQ ID NOS 212 to 214.
  • von Willebrand Factor specific bind aptamers for use as therapeutics and/or diagnostics include any one of the following sequences: ARC1029 (SEQ ID NO 214), ARC115 (SEQ ID NO 221), ARC1172 (SEQ ID NO 222), ARC1346 (SEQ ID NO 281), ARC1361 (SEQ ID NO 284), ARC1368 (SEQ ID NO 291), ARC1635 (SEQ ID NO 319), ARC1759 (SEQ ID NO 318), ARC1779 (SEQ ID NO 320), ARC1780 (SEQ ID NO 321), ARC1884 (SEQ ID NO 322) to ARC1885 (SEQ ID NO 323).
  • aptamers of the invention that bind von Willebrand Factor are described below in Examples 1 and 2.
  • aptamers may include modifications as described herein including, e.g., conjugation to lipophilic or high molecular weight compounds (e.g., PEG), incorporation of a capping moiety, incorporation of modified nucleotides, and phosphate back bone modification (including incorporation of phosphorothioate into the phosphate backbone).
  • an isolated, non-naturally occurring aptamer that binds to von Willebrand Factor is provided.
  • the aptamer of the invention modulates a function of von Willebrand Factor.
  • the aptamer of the invention inhibits a function of von Willebrand Factor while in another embodiment the aptamer stimulates a function of von Willebrand Factor.
  • the aptamer binds and/or modulates a function of a von Willebrand Factor variant.
  • a von Willebrand Factor variant as used herein encompasses variants that perform essentially the same function as a von Willebrand Factor function, preferably comprises substantially the same structure and in some embodiments comprises at least 70% sequence identity, preferably at least 80% sequence identity, more preferably at least 90% sequence identity, and more preferably at least 95% sequence identity to the amino acid sequence of human von Willebrand Factor.
  • the aptamer has substantially the same ability to bind von Willebrand Factor as that of an aptamer comprising any one of SEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98 to 164, SEQ ID NO 165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS 212-214, ARC1115, ARC1172 (SEQ ID NO 222) (SEQ ID NO 222), ARC1194 (SEQ ID NO 223) to ARC1240 (SEQ ID NO 269), ARC1338 (SEQ ID NO 273) to ARC1346 (SEQ ID NO 281), ARC1361 (SEQ ID NO
  • the aptamer has substantially the same structure and ability to bind von Willebrand Factor as that of an aptamer comprising any one of SEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98 to 165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS 212-214, ARC1115, ARC1172 (SEQ ID NO 222) (SEQ ID NO 222), ARC1194 (SEQ ID NO 223) to ARC1240 (SEQ ID NO 269), ARC1338 (SEQ ID NO 273) to ARC1346 (SEQ ID NO 281), ARC1361 (SEQ ID NO 284) to ARC
  • the aptamers of the invention comprise a sequence according to any one of SEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98 to 165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS 212-213, ARC1115, ARC1172 (SEQ ID NO 222) (SEQ ID NO 222), ARC1194 (SEQ ID NO 223) to ARC1240 (SEQ ID NO 269), ARC1338 (SEQ ID NO 273) to ARC1346 (SEQ ID NO 281), ARC1361 (SEQ ID NO 284) to ARC1381 (SEQ ID NO 304), ARC1524 (SEQ ID NO 169, S
  • the aptamers of the invention comprise a sequence that is at least 80% identical, preferably at least 90% identical and in some embodiments at least 95% identical to a sequence according to any one of SEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98 to 164, SEQ ID NO 165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS 212-214, ARC1115, ARC1172 (SEQ ID NO 222) (SEQ ID NO 222), ARC1194 (SEQ ID NO 223) to ARC1240 (SEQ ID NO 269), ARC1338 (SEQ ID NO 273) to ARC1346 (SEQ ID NO 281)
  • the aptamers of the invention specifically bind von Willebrand Factor and comprise a sequence of 30 contiguous nucleotides that are identical to 30 contiguous nucleotides in any one of the aptamers selected from the group consisting of: SEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98 to 164, SEQ ID NO 165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS 212-214, ARC1115, ARC1172 (SEQ ID NO 222) (SEQ ID NO 222), ARC1194 (SEQ ID NO 223) to ARC1240 (SEQ ID NO 269), ARC1338 (S
  • the aptamers of the invention are used as an active ingredient in pharmaceutical compositions.
  • the aptamers of the invention or compositions comprising the aptamers of the invention are used to treat thrombotic disease such as cardiovascular disorders, including acute coronary syndrome; peripheral arterial disease; and cerebrovascular disorders, including stroke.
  • the aptamers of the invention or compositions comprising the aptamers of the invention are use to treat, prevent or ameliorate a disorder selected from the group consisting of: essential thrombocytopenia: thrombotic thrombocopenic purpura (“TTP”), Type IIb von Willebrand's disease, pseudo von Willebrand disease, peripheral artery disease, e.g.
  • TTP thrombotic thrombocopenic purpura
  • peripheral arterial occlusive disease unstable angina, angina pectoris, arterial thrombosis, atherosclerosis, myocardial infarction, acute coronary syndrome, atrial fibrillation, carotid stenosis, cerebral infarction, cerebral thrombosis, ischemic stroke, and transient cerebral ischemic attack.
  • the pharmaceutical composition of the invention is administered prior to/during and/or after dialysis, CABG surgery, percutaneous coronary intervention or heart valve replacement.
  • aptamer therapeutics of the present invention have great affinity and specificity to their targets while reducing the deleterious side effects from non-naturally occurring nucleotide substitutions if the aptamer therapeutics break down in the body of patients or subjects.
  • the therapeutic compositions containing the aptamer therapeutics of the present invention are free of or have a reduced amount of fluorinated nucleotides.
  • the aptamers of the present invention can be synthesized using any oligonucleotide synthesis techniques known in the art including solid phase oligonucleotide synthesis techniques (see, e.g., Froehler et al., Nucl. Acid Res. 14:5399-5467 (1986) and Froehler et al., Tet. Lett. 27:5575-5578 (1986)) and solution phase methods such as triester synthesis methods (see, e.g., Sood et al., Nucl. Acid Res. 4:2557 (1977) and Hirose et al., Tet. Lett, 28:2449 (1978)) both of which are well known in the art.
  • solid phase oligonucleotide synthesis techniques see, e.g., Froehler et al., Nucl. Acid Res. 14:5399-5467 (1986) and Froehler et al., Tet.
  • the invention also includes pharmaceutical compositions containing aptamer molecules that bind to von Willebrand Factor.
  • the compositions are suitable for internal use and include an effective amount of a pharmacologically active compound of the invention, alone or in combination, with one or more pharmaceutically acceptable carriers.
  • the compounds are especially useful in that they have very low, if any toxicity.
  • compositions of the invention can be used to treat or prevent a pathology, such as a disease or disorder, or alleviate the symptoms of such disease or disorder in a patient.
  • a pathology such as a disease or disorder
  • compositions of the present invention can be used to treat or prevent a pathology associated with platelet aggregation.
  • the disease to be treated, prevented or ameliorated is selected from the group consisting of: essential thrombocytopenia: thrombotic thrombocopenic purpura (“TTP”), Type IIb von Willebrand's disease, pseudo von Willebrand disease, peripheral artery disease, e.g.
  • peripheral arterial occlusive disease unstable angina, angina pectoris, arterial thrombosis, atherosclerosis, myocardial infarction, acute coronary syndrome, atrial fibrillation, carotid stenosis, cerebral infarction, cerebral thrombosis, ischemic stroke, and transient cerebral ischemic attack.
  • the pharmaceutical composition of the invention is administered prior to, during and/or after dialysis, CABG surgery, percutaneous coronary intervention or heart valve replacement.
  • compositions of the invention are useful for administration to a subject suffering from, or predisposed to, a disease or disorder which is related to or derived from a target to which the aptamers of the invention specifically bind.
  • compositions of the invention can be used in a method for treating a patient or subject having a pathology.
  • the method involves administering to the patient or subject an aptamer or a composition comprising aptamers that bind to von Willebrand Factor involved with the pathology, so that binding of the aptamer to the target alters the biological function of von Willebrand Factor, thereby treating the pathology.
  • the patient or subject having a pathology i.e., the patient or subject treated by the methods of this invention can be a mammal, more particularly a vertebrate, or more particularly, a human.
  • the aptamers or their pharmaceutically acceptable salts are administered in amounts which will be sufficient to exert their desired biological activity, e.g., preventing vWF dependent platelet aggregation.
  • One aspect of the invention comprises an aptamer composition of the invention in combination with other treatments for thrombotic related disorders.
  • the aptamer composition of the invention may contain, for example, more than one aptamer, e.g. an anti-thrombin aptamer and an anti-vWF aptamer.
  • an aptamer composition of the invention, containing one or more aptamers of the invention is administered in combination with another useful composition such as an anti-inflammatory agent, an immunosuppressant, an antiviral agent, or the like.
  • another useful composition such as an anti-inflammatory agent, an immunosuppressant, an antiviral agent, or the like.
  • the currently available dosage forms of the known therapeutic agents for use in such combinations will be suitable.
  • Combination therapy includes the administration of an aptamer composition of the invention and at least a second agent as part of a specific treatment regimen intended to provide the beneficial effect from the co-action of these therapeutic agents.
  • the beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents.
  • Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually minutes, hours, days or weeks depending upon the combination selected).
  • “Combination therapy” may, but generally is not, intended to encompass the administration of two or more of these therapeutic agents as part of separate monotherapy regimens that incidentally and arbitrarily result in the combinations of the present invention. “Combination therapy” is intended to embrace administration of these therapeutic agents in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous manner. Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single capsule having a fixed ratio of each therapeutic agent or in multiple, single capsules for each of the therapeutic agents.
  • Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, topical routes, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues.
  • the therapeutic agents can be administered by the same route or by different routes.
  • a first therapeutic agent of the combination selected may be administered by injection while the other therapeutic agents of the combination may be administered topically.
  • Combination therapy also can embrace the administration of the therapeutic agents as described above in further combination with other biologically active ingredients.
  • the combination therapy further comprises a non-drug treatment
  • the non-drug treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and non-drug treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the non-drug treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.
  • Therapeutic or pharmacological compositions of the present invention will generally comprise an effective amount of the active component(s) of the therapy, dissolved or dispersed in a pharmaceutically acceptable medium.
  • Pharmaceutically acceptable media or carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the therapeutic compositions of the present invention.
  • compositions will be known to those of skill in the art in light of the present disclosure.
  • such compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection; as tablets or other solids for oral administration; as time release capsules; or in any other form currently used, including eye drops, creams, lotions, salves, inhalants and the like.
  • injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection; as tablets or other solids for oral administration; as time release capsules; or in any other form currently used, including eye drops, creams, lotions, salves, inhalants and the like.
  • sterile formulations such as saline-based washes, by surgeons, physicians or health care workers to treat a particular area in the operating field may also be particularly useful.
  • Compositions may also be delivered via microdevice, microparticle or sponge.
  • therapeutics Upon formulation, therapeutics will be administered in a manner compatible with the dosage formulation, and in such amount as is pharmacologically effective.
  • the formulations are easily administered in a variety of dosage forms.
  • the aptamer of the invention is formulated as an injectable solution described above, but drug release capsules and the like can also be employed.
  • the quantity of active ingredient and volume of composition to be administered depends on the host animal to be treated. Precise amounts of active compound required for administration depend on the judgment of the practitioner and are peculiar to each individual.
  • a minimal volume of a composition required to disperse the active compounds is typically utilized. Suitable regimes for administration are also variable, but would be typified by initially administering the compound and monitoring the results and then giving further controlled doses at further intervals.
  • the effects of administration of the anti-vWF aptamer of the invention could be monitored by measuring platelet aggregation formation such as measuring botrocetin induced platelet aggregation (“BIPA”) and/or shear force induced hemostatic plug formation using the PFA-100 instrument as described in Example 3 below.
  • BIPA botrocetin induced platelet aggregation
  • the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture.
  • Suitable binders include starch, magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, polyethylene glycol, waxes and the like.
  • Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol and the like.
  • Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum starches, agar, alginic acid or its sodium salt, or effervescent mixtures, and the like.
  • Diluents include, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine.
  • compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions.
  • the compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances.
  • the compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and typically contain about 0.1 to 75%, preferably about 1 to 50%, of the active ingredient.
  • the compounds of the invention can also be administered in such oral dosage forms as timed release and sustained release tablets or capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions.
  • Liquid, particularly injectable compositions can, for example, be prepared by dissolving, dispersing, etc.
  • the active compound is dissolved in or mixed with a pharmaceutically pure solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form the injectable solution or suspension.
  • a pharmaceutically pure solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like.
  • solid forms suitable for dissolving in liquid prior to injection can be formulated.
  • the compounds of the present invention can be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
  • injectables can be prepared in conventional forms, either as liquid solutions or suspensions.
  • Parenteral injectable administration is generally used for subcutaneous, intramuscular or intravenous injections and infusions. Additionally, one approach for parenteral administration employs the implantation of a slow-release or sustained-released systems, which assures that a constant level of dosage is maintained, according to U.S. Pat. No. 3,710,795, incorporated herein by reference.
  • preferred compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, inhalants, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
  • the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
  • Other preferred topical preparations include creams, ointments, lotions, aerosol sprays and gels, wherein the concentration of active ingredient would typically range from 0.01% to 15%, w/w or w/v.
  • excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like may be used.
  • the active compound defined above may be also formulated as suppositories using for example, polyalkylene glycols, for example, propylene glycol, as the carrier.
  • suppositories are advantageously prepared from fatty emulsions or suspensions.
  • the compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, containing cholesterol, stearylamine or phosphatidylcholines.
  • a film of lipid components is hydrated with an aqueous solution of drug to a form lipid layer encapsulating the drug, as described in U.S. Pat. No. 5,262,564.
  • the aptamer molecules described herein can be provided as a complex with a lipophilic compound or non-immunogenic, high molecular weight compound constructed using methods known in the art.
  • liposomes may bear aptamers on their surface for targeting and carrying cytotoxic agents internally to mediate cell killing.
  • nucleic-acid associated complexes is provided in U.S. Pat. No. 6,011,020.
  • the compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers.
  • soluble polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl-methacrylamide-phenol, polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues.
  • the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
  • the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as for example, sodium acetate, and triethanolamine oleate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as for example, sodium acetate, and triethanolamine oleate.
  • the dosage regimen utilizing the aptamers is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular aptamer or salt thereof employed.
  • An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
  • Oral dosages of the present invention when used for the indicated effects, will range between about 0.05 to 7500 mg/day orally.
  • the compositions are preferably provided in the form of scored tablets containing 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100.0, 250.0, 500.0 and 1000.0 mg of active ingredient.
  • Infused dosages, intranasal dosages and transdermal dosages will range between 0.05 to 7500 mg/day.
  • Subcutaneous, intravenous and intraperitoneal dosages will range between 0.05 to 3800 mg/day.
  • Compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.
  • Effective plasma levels of the compounds of the present invention range from 0.002 mg/mL to 50 mg/mL.
  • mass refers only to the molecular weight of the oligonucleotide portion of the aptamer, irrespective of the mass conferred by PEG conjugation.
  • aptamers oligonucleotide-based therapeutics, including aptamers, be tailored to match the desired pharmaceutical application. While aptamers directed against extracellular targets do not suffer from difficulties associated with intracellular delivery (as is the case with antisense and RNAi-based therapeutics), such aptamers must still be able to be distributed to target organs and tissues, and remain in the body (unmodified) for a period of time consistent with the desired dosing regimen.
  • the present invention provides materials and methods to affect the pharmacokinetics of aptamer compositions, and, in particular, the ability to tune aptamer pharmacokinetics.
  • the tunability of (i.e., the ability to modulate) aptamer pharmacokinetics is achieved through conjugation of modifying moieties (e.g., PEG polymers) to the aptamer and/or the incorporation of modified nucleotides (e.g., 2′-fluoro or 2′-O-methyl) to alter the chemical composition of the nucleic acid.
  • modifying moieties e.g., PEG polymers
  • modified nucleotides e.g., 2′-fluoro or 2′-O-methyl
  • aptamers in circulation it is desirable to decrease the residence times of aptamers in the circulation.
  • maintenance therapies where systemic circulation of a therapeutic is desired, it may be desirable to increase the residence times of aptamers in circulation.
  • the tunability of aptamer pharmacokinetics is used to modify the biodistribution of an aptamer therapeutic in a subject.
  • the aptamer therapeutic preferentially accumulates in a specific tissue or organ(s).
  • PEGylation of an aptamer therapeutic e.g., PEGylation with a 20 kDa PEG polymer
  • PEGylation with a 20 kDa PEG polymer is used to target inflamed tissues, such that the PEGylated aptamer therapeutic preferentially accumulates in inflamed tissue.
  • aptamer therapeutics e.g., aptamer conjugates or aptamers having altered chemistries, such as modified nucleotides
  • parameters include, for example, the half-life (t 1/2 ), the plasma clearance (CL), the volume of distribution (Vss), the area under the concentration-time curve (AUC), maximum observed serum or plasma concentration (C max ), and the mean residence time (MRT) of an aptamer composition.
  • AUC refers to the area under the plot of the plasma concentration of an aptamer therapeutic versus the time after aptamer administration.
  • the AUC value is used to estimate the bioavailability (i.e., the percentage of administered aptamer therapeutic in the circulation after aptamer administration) and/or total clearance (CL) (i.e., the rate at which the aptamer therapeutic is removed from circulation) of a given aptamer therapeutic.
  • the volume of distribution relates the plasma concentration of an aptamer therapeutic to the amount of aptamer present in the body. The larger the Vss, the more an aptamer is found outside of the plasma (i.e., the more extravasation).
  • the present invention provides materials and methods to modulate, in a controlled manner, the pharmacokinetics and biodistribution of stabilized aptamer compositions in vivo by conjugating an aptamer to a modulating moiety such as a small molecule, peptide, or polymer terminal group, or by incorporating modified nucleotides into an aptamer.
  • a modulating moiety such as a small molecule, peptide, or polymer terminal group
  • conjugation of a modifying moiety and/or altering nucleotide(s) chemical composition alter fundamental aspects of aptamer residence time in circulation and distribution to tissues.
  • oligonucleotide therapeutics are subject to elimination via renal filtration.
  • a nuclease-resistant oligonucleotide administered intravenously typically exhibits an in vivo half-life of ⁇ 10 min, unless filtration can be blocked. This can be accomplished by either facilitating rapid distribution out of the blood stream into tissues or by increasing the apparent molecular weight of the oligonucleotide above the effective size cut-off for the glomerulus.
  • Conjugation of small therapeutics to a PEG polymer (PEGylation), described below, can dramatically lengthen residence times of aptamers in circulation, thereby decreasing dosing frequency and enhancing effectiveness against vascular targets.
  • Aptamers can be conjugated to a variety of modifying moieties, such as high molecular weight polymers, e.g., PEG; peptides, e.g., Tat (a 13-amino acid fragment of the HIV Tat protein (Vives, et al., (1997), J. Biol. Chem.
  • Ant a 16-amino acid sequence derived from the third helix of the Drosophila antennapedia homeotic protein (Pietersz, et al., (2001), Vaccine 19(11-12): 1397-405)
  • Arg 7 a short, positively charged cell-permeating peptides composed of polyarginine (Arg 7 ) (Rothbard, et al., (2000), Nat. Med. 6(11): 1253-7; Rothbard, J et al., (2002), J. Med. Chem. 45(17): 3612-8)); and small molecules, e.g., lipophilic compounds such as cholesterol.
  • aptamers in vivo properties of aptamers are altered most profoundly by complexation with PEG groups.
  • complexation of a mixed 2′ F. and 2′-OMe modified aptamer therapeutic with a 20 kDa PEG polymer hinders renal filtration and promotes aptamer distribution to both healthy and inflamed tissues.
  • the 20 kDa PEG polymer-aptamer conjugate proves nearly as effective as a 40 kDa PEG polymer in preventing renal filtration of aptamers.
  • the prolonged systemic exposure afforded by presence of the 20 kDa moiety also facilitates distribution of aptamer to tissues, particularly those of highly perfused organs and those at the site of inflammation.
  • the aptamer-20 kDa PEG polymer conjugate directs aptamer distribution to the site of inflammation, such that the PEGylated aptamer preferentially accumulates in inflamed tissue.
  • the 20 kDa PEGylated aptamer conjugate is able to access the interior of cells, such as, for example, kidney cells.
  • Modified nucleotides can also be used to modulate the plasma clearance of aptamers.
  • an unconjugated aptamer which incorporates both 2′-F and 2′-OMe stabilizing chemistries, which is typical of current generation aptamers as it exhibits a high degree of nuclease stability in vitro and in vivo, displays rapid loss from plasma (i.e., rapid plasma clearance) and a rapid distribution into tissues, primarily into the kidney, when compared to unmodified aptamer.
  • nucleic acids with high molecular weight non-immunogenic polymers has the potential to alter the pharmacokinetic and pharmacodynamic properties of nucleic acids making them more effective therapeutic agents.
  • Favorable changes in activity can include increased resistance to degradation by nucleases, decreased filtration through the kidneys, decreased exposure to the immune system, and altered distribution of the therapeutic through the body.
  • the aptamer compositions of the invention may be derivatized with polyalkylene glycol (PAG) moieties.
  • PAG polyalkylene glycol
  • PAG-derivatized nucleic acids are found in United States patent application Ser. No. 10/718,833, filed on Nov. 21, 2003, which is herein incorporated by reference in its entirety.
  • Typical polymers used in the invention include poly(ethylene glycol) (PEG), also known as poly(ethylene oxide) (PEO) and polypropylene glycol (including poly isopropylene glycol). Additionally, random or block copolymers of different alkylene oxides (e.g., ethylene oxide and propylene oxide) can be used in many applications.
  • a polyalkylene glycol such as PEG
  • PEG is a linear polymer terminated at each end with hydroxyl groups: HO—CH 2 CH 2 O—(CH 2 CH 2 O) n —CH 2 CH 2 —OH.
  • This polymer, alpha-, omega-dihydroxylpoly(ethylene glycol) can also be represented as HO-PEG-OH, where it is understood that the -PEG-symbol represents the following structural unit: —CH 2 CH 2 O—(CH 2 CH 2 O) n —CH 2 CH 2 — where n typically ranges from about 4 to about 10,000.
  • the PEG molecule is di-functional and is sometimes referred to as “PEG diol.”
  • the terminal portions of the PEG molecule are relatively non-reactive hydroxyl moieties, the —OH groups, that can be activated, or converted to functional moieties, for attachment of the PEG to other compounds at reactive sites on the compound.
  • Such activated PEG diols are referred to herein as bi-activated PEGs.
  • the terminal moieties of PEG diol have been functionalized as active carbonate ester for selective reaction with amino moieties by substitution of the relatively nonreactive hydroxyl moieties, —OH, with succinimidyl active ester moieties from N-hydroxy succinimide.
  • PEG molecule on one end it is desirable to cap the PEG molecule on one end with an essentially non-reactive moiety so that the PEG molecule is mono-functional (or mono-activated).
  • bi-functional activated PEGs lead to extensive cross-linking, yielding poorly functional aggregates.
  • one hydroxyl moiety on the terminus of the PEG diol molecule typically is substituted with non-reactive methoxy end moiety, —OCH 3 .
  • the other, un-capped terminus of the PEG molecule typically is converted to a reactive end moiety that can be activated for attachment at a reactive site on a surface or a molecule such as a protein.
  • PAGs are polymers which typically have the properties of solubility in water and in many organic solvents, lack of toxicity, and lack of immunogenicity.
  • One use of PAGs is to covalently attach the polymer to insoluble molecules to make the resulting PAG-molecule “conjugate” soluble.
  • the water-insoluble drug paclitaxel when coupled to PEG, becomes water-soluble. Greenwald, et al., J. Org. Chem., 60:331-336 (1995).
  • PAG conjugates are often used not only to enhance solubility and stability but also to prolong the blood circulation half-life of molecules.
  • Polyalkylated compounds of the invention are typically between 5 and 80 kDa in size however any size can be used, the choice dependent on the aptamer and application.
  • Other PAG compounds of the invention are between 10 and 80 kDa in size.
  • Still other PAG compounds of the invention are between 10 and 60 kDa in size.
  • a PAG polymer may be at least 10, 20, 30, 40, 50, 60, or 80 kDa in size. Such polymers can be linear or branched.
  • nucleic acid therapeutics are typically chemically synthesized from activated monomer nucleotides.
  • PEG-nucleic acid conjugates may be prepared by incorporating the PEG using the same iterative monomer synthesis.
  • PEGs activated by conversion to a phosphoramidite form can be incorporated into solid-phase oligonucleotide synthesis.
  • oligonucleotide synthesis can be completed with site-specific incorporation of a reactive PEG attachment site. Most commonly this has been accomplished by addition of a free primary amine at the 5′-terminus (incorporated using a modifier phosphoramidite in the last coupling step of solid phase synthesis).
  • a reactive PEG e.g., one which is activated so that it will react and form a bond with an amine
  • the coupling reaction is carried out in solution.
  • the ability of PEG conjugation to alter the biodistribution of a therapeutic is related to a number of factors including the apparent size (e.g., as measured in terms of hydrodynamic radius) of the conjugate. Larger conjugates (>10 kDa) are known to more effectively block filtration via the kidney and to consequently increase the serum half-life of small macromolecules (e.g., peptides, antisense oligonucleotides). The ability of PEG conjugates to block filtration has been shown to increase with PEG size up to approximately 50 kDa (further increases have minimal beneficial effect as half life becomes defined by macrophage-mediated metabolism rather than elimination via the kidneys).
  • small macromolecules e.g., peptides, antisense oligonucleotides
  • Branched activated PEGs will have more than two termini, and in cases where two or more termini have been activated, such activated higher molecular weight PEG molecules are referred to herein as, multi-activated PEGs. In some cases, not all termini in a branch PEG molecule are activated. In cases where any two termini of a branch PEG molecule are activated, such PEG molecules are referred to as bi-activated PEGs. In some cases where only one terminus in a branch PEG molecule is activated, such PEG molecules are referred to as mono-activated.
  • the present invention provides another cost effective route to the synthesis of high molecular weight PEG-nucleic acid (preferably, aptamer) conjugates including multiply PEGylated nucleic acids.
  • PEG-nucleic acid preferably, aptamer
  • the present invention also encompasses PEG-linked multimeric oligonucleotides, e.g., dimerized aptamers.
  • the present invention also relates to high molecular weight compositions where a PEG stabilizing moiety is a linker which separates different portions of an aptamer, e.g., the PEG is conjugated within a single aptamer sequence, such that the linear arrangement of the high molecular weight aptamer composition is, e.g., nucleic acid -PEG-nucleic acid (-PEG-nucleic acid) n where n is greater than or equal to 1.
  • a PEG stabilizing moiety is a linker which separates different portions of an aptamer, e.g., the PEG is conjugated within a single aptamer sequence, such that the linear arrangement of the high molecular weight aptamer composition is, e.g., nucleic acid -PEG-nucleic acid (-PEG-nucleic acid) n where n is greater than or equal to 1.
  • High molecular weight compositions of the invention include those having a molecular weight of at least 10 kDa. Compositions typically have a molecular weight between 10 and 80 kDa in size. High molecular weight compositions of the invention are at least 10, 20, 30, 40, 50, 60, or 80 kDa in size.
  • a stabilizing moiety is a molecule, or portion of a molecule, which improves pharmacokinetic and pharmacodynamic properties of the high molecular weight aptamer compositions of the invention.
  • a stabilizing moiety is a molecule or portion of a molecule which brings two or more aptamers, or aptamer domains, into proximity, or provides decreased overall rotational freedom of the high molecular weight aptamer compositions of the invention.
  • a stabilizing moiety can be a polyalkylene glycol, such a polyethylene glycol, which can be linear or branched, a homopolymer or a heteropolymer.
  • Other stabilizing moieties include polymers such as peptide nucleic acids (PNA).
  • Oligonucleotides can also be stabilizing moieties; such oligonucleotides can include modified nucleotides, and/or modified linkages, such as phosphorothioates.
  • a stabilizing moiety can be an integral part of an aptamer composition, i.e., it is covalently bonded to the aptamer.
  • compositions of the invention include high molecular weight aptamer compositions in which two or more nucleic acid moieties are covalently conjugated to at least one polyalkylene glycol moiety.
  • the polyalkylene glycol moieties serve as stabilizing moieties.
  • the primary structure of the covalent molecule includes the linear arrangement nucleic acid-PAG-nucleic acid.
  • One example is a composition having the primary structure nucleic acid-PEG-nucleic acid.
  • Another example is a linear arrangement of: nucleic acid-PEG-nucleic acid-PEG-nucleic acid.
  • the nucleic acid is originally synthesized such that it bears a single reactive site (e.g., it is mono-activated).
  • this reactive site is an amino group introduced at the 5′-terminus by addition of a modifier phosphoramidite as the last step in solid phase synthesis of the oligonucleotide.
  • a modifier phosphoramidite as the last step in solid phase synthesis of the oligonucleotide.
  • the concentration of oligonucleotide is 1 mM and the reconstituted solution contains 200 mM NaHCO 3 -buffer, pH 8.3.
  • Synthesis of the conjugate is initiated by slow, step-wise addition of highly purified bi-functional PEG.
  • the PEG diol is activated at both ends (bi-activated) by derivatization with succinimidyl propionate.
  • the PEG-nucleic acid conjugate is purified by gel electrophoresis or liquid chromatography to separate fully-, partially-, and un-conjugated species.
  • Multiple PAG molecules concatenated (e.g., as random or block copolymers) or smaller PAG chains can be linked to achieve various lengths (or molecular weights).
  • Non-PAG linkers can be used between PAG chains of varying lengths.
  • the 2′-O-methyl, 2′-fluoro and other modified nucleotide modifications stabilize the aptamer against nucleases and increase its half life in vivo.
  • the 3′-3′-dT cap also increases exonuclease resistance. See, e.g., U.S. Pat. Nos. 5,674,685; 5,668,264; 6,207,816; and 6,229,002, each of which is incorporated by reference herein in its entirety.
  • High molecular weight PAG-nucleic acid-PAG conjugates can be prepared by reaction of a mono-functional activated PEG with a nucleic acid containing more than one reactive site.
  • the nucleic acid is bi-reactive, or bi-activated, and contains two reactive sites: a 5′-amino group and a 3′-amino group introduced into the oligonucleotide through conventional phosphoramidite synthesis, for example: 3′-5′-di-PEGylation as illustrated in FIG. 2 .
  • reactive sites can be introduced at internal positions, using for example, the 5-position of pyrimidines, the 8-position of purines, or the 2′-position of ribose as sites for attachment of primary amines.
  • the nucleic acid can have several activated or reactive sites and is said to be multiply activated.
  • the modified oligonucleotide is combined with the mono-activated PEG under conditions that promote selective reaction with the oligonucleotide reactive sites while minimizing spontaneous hydrolysis.
  • monomethoxy-PEG is activated with succinimidyl propionate and the coupled reaction is carried out at pH 8.3.
  • PEG-nucleic acid conjugate is purified by gel electrophoresis or liquid chromatography to separate fully-, partially-, and un-conjugated species.
  • the linking domains can also have one or more polyalkylene glycol moieties attached thereto.
  • PAGs can be of varying lengths and may be used in appropriate combinations to achieve the desired molecular weight of the composition.
  • linker The effect of a particular linker can be influenced by both its chemical composition and length.
  • a linker that is too long, too short, or forms unfavorable steric and/or ionic interactions with the target will preclude the formation of complex between aptamer and target.
  • a linker, which is longer than necessary to span the distance between nucleic acids, may reduce binding stability by diminishing the effective concentration of the ligand. Thus, it is often necessary to optimize linker compositions and lengths in order to maximize the affinity of an aptamer to a target.
  • a DNA template with the sequence 5′-GGAGCGCACTCAGCCAC NNNNNNNTTTCGACCTCTCTGCTAGC 3′ (SEQ ID NO 8) was synthesized using an ABI EXPEDITETM DNA synthesizer, and deprotected by standard methods.
  • the series of N's in the DNA template (SEQ ID NO 8) can be any combination of nucleotides and gives rise to the unique sequence region of the resulting aptamers.
  • the template was amplified with the primers 5′-TAATACGACTCACTATAGGAGCGCACTCAGCCAC-3′ (SEQ ID NO 9) and 5′-GCTAGCAGAGAGGTCGAAA-3′ (SEQ ID NO 10) and then used as a template for in vitro transcription with T7 RNA polymerase (Y639F). Transcriptions were, typically, incubated at 37° C.
  • the first ten rounds were initiated by immobilizing 24 pmoles of human vWF A1 domain (SEQ ID NO 4, FIG. 4 ) to the surface of a Nunc Maxisorp hydrophobic plate (Nunc Cat. #446612, Rochester, N.Y.) for 1 hour at room temperature in 100 ⁇ L of 1 ⁇ Dulbecco's PBS (Gibco BRL Cat. #14040-133, Carlsbad, Calif.).
  • For Rounds eleven and twelve, 12 pmoles of full length human vWF (SEQ ID NO 7, accession number VWHU, available from Calbiochem Cat. #681300, La Jolla, Calif.) were immobilized to the hydrophobic plate.
  • each round was initiated by immobilizing 24 pmoles of rabbit vWF A1 domain (SEQ ID NO 6: accession number AAB51555, FIG. 3 ) under the same conditions as for human vWF A1 domain.
  • the RNA was transferred to a BSA blocked well (not containing the protein target) for 1 hour at room temperature to remove any BSA binding sequences from the pool prior to the positive selection.
  • 0.1 mg/mL tRNA and 0.1 mg/mL salmon sperm DNA were spiked into the positive selection reaction as non-specific competitors.
  • RNA bound to the immobilized protein target was reverse transcribed directly in the selection plate with the addition of RT mix (Round 1: 100 uL; Round 2+: 50 uL; containing the 3′-primer according to SEQ ID NO 10 and Thermoscript RT (Invitrogen Cat. #11146-016, Carlsbad, Calif.) followed by incubation at 65° C. for 1 hour.
  • PCR reactions were done under the following conditions: a) denaturation step: 94° C. for 2 minutes; b) cycling steps: 94° C. for 30 seconds, 60° C. for 30 seconds, 72° C. for 1 minute; c) final extension step: 72° C. for 3 minutes. The cycles were repeated until sufficient PCR product was generated.
  • the minimum number of cycles required to generate sufficient PCR product is reported in Tables 1 and 2 as the “PCR Threshold”.
  • the amplified pool template DNA was then isopropanol precipitated and half of the PCR product was used as template for the transcription of pool RNA for the next round of selection.
  • the transcribed RNA pool was gel purified using a 10% polyacrylamide gel every third round. When not gel-purified, the transcribed RNA pool was desalted using two Centri-Spin 10 columns (Princeton Separations Cat. #CS-101, Adelphia, N.J.). In all cases, an equivalent of one-tenth of the total transcription product was carried forward as the starting pool for the subsequent round of selection.
  • the selection progress was monitored using a sandwich filter binding assay.
  • the 5′- 32 P-labeled pool RNA (trace concentration) was incubated with either a no target protein control, 100 nM human vWF A1 domain (SEQ ID NO 4) or 100 nM rabbit vWF A1 domain (SEQ ID NO 6) in 1 ⁇ Dulbecco's PBS containing 0.1 mg/mL tRNA, and 0.1 mg/mL salmon sperm DNA (in a final volume of 50 uL) for 30 minutes at room temperature and then applied to a nitrocellulose and nylon filter sandwich in a dot blot apparatus (Schleicher and Schuell, Keene, N.H.).
  • the percentage of pool RNA bound to the nitrocellulose was calculated after Rounds 6, 9, and 12 with a three point screen (100 nM human vWF A1 domain, 100 nM rabbit vWF A1 domain, and a no-target control). Pool binding was compared to that of the na ⁇ ve pool RNA (Round 0). The results of the rRfY pool binding analyses are found in Table 3.
  • RNA in the presence of human or rabbit vWF A1 domain versus in the absence of protein was seen, the pools were cloned using the TOPO TA cloning kit (Invitrogen, Cat. #45-0641, Carlsbad, Calif.) according to the manufacturer's instructions. Round 9 and 12 pool templates were cloned and sequenced (125 total sequences), producing 48 unique clones. All unique clones were transcribed, desalted, 5- 32 P end-labeled, and assayed in a 3-point dot blot screen (no protein target control, 100 nM human vWF A1 domain (SEQ ID NO 5, FIG.
  • K D s were determined for 12 of the best vWF dependent binding sequences using the dot blot assay and are reported in column 5 of Table 4 below.
  • aptamers transcripts were purified on 10% denaturing polyacrylamide gels, 5′ end labeled with ⁇ - 32 P ATP.
  • human vWF A1 domain is diluted with 1 ⁇ Dulbecco's PBS buffer which includes 0.1 mg/mL BSA and incubated with labeled aptamer for 30 minutes at 24° C. prior to filtration and quantitation.
  • nucleic acid sequences of the rRfY aptamers characterized in Table 4 above are given below.
  • the unique sequence of each aptamer below begins at nucleotide 18, immediately following the sequence GGAGCGCACTCAGCCAC (SEQ ID NO 221), and runs until it meets the 3′fixed nucleic acid sequence TTTCGACCTCTCTGCTAGC (SEQ ID NO 222).
  • ARC840 SEQ ID NO 23 is one example of an aptamer having the sequence depicted in FIG. 10 wherein the bold, underlined regions shown in the sequence listed above denote required bases.
  • aptamers that bind to (1) human vWF A1 domain, (2) rabbit vWF A1 domain, or (3) human and rabbit vWF A1 domains using a nucleotide pool consisting of 2′-OH purine and deoxy-pyrimidine nucleotides (rRdY).
  • the selection strategy yielded high affinity aptamers specific for human and rabbit vWF A1 domains which had been immobilized on a hydrophobic plate.
  • a DNA template with the sequence 5′-GGAGCGCACTCAGCCAC NNNNNNNTTTCGACCTCTCTGCTAGC 3′ (SEQ ID NO 8) was synthesized using an ABI EXPEDITETM DNA synthesizer, and deprotected by standard methods.
  • the series of N's in the DNA template (SEQ ID NO 8) can be any combination of nucleotides and gives rise to the unique sequence region of the resulting aptamers.
  • the template was amplified with the primers 5′-TAATACGACTCACTATAGGAGCGCACTCAGCCAC-3′ (SEQ ID NO 9) and 5′-GCTAGCAGAGAGGTCGAAA-3′ (SEQ ID NO 10) and then used as a template for in vitro transcription with T7 RNA polymerase (Y639F). Transcriptions were, typically, incubated at 37° C.
  • each round was initiated by immobilizing 24 pmoles of rabbit vWF A1 domain (SEQ ID NO 6) under the same conditions.
  • 12 pmoles of human vWF A1 domain (SEQ ID NO 4) and 12 pmoles of rabbit vWF A1 domain (SEQ ID NO 6) were immobilized to a hydrophobic plate as previously described.
  • the protein target was alternated each round between the human and rabbit vWF A1 domain (SEQ ID NOS 4 and 5, respectively), except in round 11, human full length vWF (SEQ ID NO 7) was used.
  • the RNA was transferred to a BSA blocked well (not containing the protein target) for 1 hour at room temperature to remove any BSA binding sequences from the pool prior to the positive selection.
  • 0.1 mg/mL tRNA and 0.1 mg/mL salmon sperm DNA were spiked into the positive selection reaction as non-specific competitors.
  • RNA bound to the immobilized protein target was reverse transcribed directly in the selection plate with the addition of RT mix (Round 1: 100 uL; Round 2+: 50 uL; containing the 3′-primer according to (SEQ ID NO 10) and Thermoscript RT (Invitrogen, Cat. #11146-016, Carlsbad, Calif.) followed by incubation at 65° C. for 1 hour.
  • PCR reactions were done under the following conditions: a) denaturation step: 94° C. for 2 minutes; b) cycling steps: 94° C. for 30 seconds, 60° C. for 30 seconds, 72° C. for 1 minute; c) final extension step: 72° C. for 3 minutes. The cycles were repeated until sufficient PCR product was generated.
  • the minimum number of cycles required to generate sufficient PCR product is reported in Tables 5, 6 and 7 as the “PCR Threshold”.
  • the amplified pool template DNA was then isopropanol precipitated and half of the PCR product was used as template for the transcription of pool RNA for the next round of selection.
  • the transcribed RNA pool was gel purified using a 10% polyacrylamide gel every two rounds. When not gel-purified, the transcribed pool was desalted using two Centri-Spin 10 columns (Princeton Separations Cat. #CS-101, Adelphia, N.J.). In all cases, an equivalent of one-tenth of the total transcription product was carried forward as the starting pool for the subsequent round of selection.
  • the selection progress was monitored using a sandwich filter binding assay.
  • the 5′- 32 P-labeled pool RNA (trace concentration) was incubated with either a no target protein control, 100 nM human vWF A1 domain or 100 nM rabbit vWF A1 domain, in 1 ⁇ Dulbecco's PBS containing 0.1 mg/mL tRNA, and 0.1 mg/mL salmon sperm DNA (in a final volume of 50 uL) for 30 minutes at room temperature and then applied to a nitrocellulose and nylon filter sandwich in a dot blot apparatus (Schleicher and Schuell, Keene, N.H.).
  • the percentage of pool RNA bound to the nitrocellulose was calculated after Rounds 6, 9, and 12 with a three point screen (100 nM human vWF A1 domain, 100 nM rabbit vWF A1 domain, and a no-target control). Pool binding was compared to that of the na ⁇ ve pool RNA (Round 0). The results of the rRdY pool binding analyses are found in Table 8.
  • RNA in the presence of human or rabbit vWF A1 domain SEQ ID NOS 4 and 6, respectively
  • the pools were cloned using the TOPO TA cloning kit (Invitrogen Cat. #45-0641, Carlsbad, Calif.) according to the manufacturer's instructions. Round 9 and 12 pool templates were cloned and sequenced (185 total sequences), producing 78 unique clones within 3 sequence families.
  • K D 'S were determined for 16 of the best vWF dependent binding sequences using the dot blot assay.
  • aptamers were purified on denaturing polyacrylamide gels and 5′-end labeled with ⁇ - 32 P ATP.
  • a 6 point protein titration of human vWF A1 domain (SEQ ID NO 5) was used in the dot blot assay (333 nM, 100 nM, 33 nM, 10 nM, 3 nM, 0 nM) in 1 ⁇ DPBS plus 0.1 mg/mL BSA at room temperature for 30 minutes.
  • nucleic acid sequences of the rRdY aptamers characterized in Table 9 above are described below.
  • the unique sequence of each aptamer below begins at nucleotide 18, immediately following the sequence GGAGCGCACTCAGCCAC (SEQ ID NO 221), and runs until it meets the 3′fixed nucleic acid sequence TTTCGACCTCTCTGCTAGC (SEQ ID NO 222).
  • the predicted secondary structure and core nucleic acid sequences required for binding to the vWF target of some embodiments of the invention is depicted in FIG. 12 as SEQ ID NO 218.
  • the predicted core nucleic acid binding motif for SEQ ID NO 49 is shown in bold and underlined below:
  • AX205.F7 (ARC 841) SEQ ID NO 49 GGAGCGCACT CAGCCACACGACATTCGCGGGTTGTAATTACCACGCATGC CTG TTTGTTTCGACCTCTCTGCTAGC (AMX205.A7), SEQ ID NO 34 GGAGCGCACTCAGCCACTCAAGGGGGTCGCGTGGGGACGAAGGGTTGCAG TGTGTCGTTTCGACCTCTCTGCTAGC
  • the predicted core nucleic acid sequences and secondary structure required for binding to the vWF target of some embodiments of the invention is depicted in FIG. 13 as SEQ ID NO 219.
  • aptamers that bind to (1) human vWF A1 domain, (2) rabbit vWF A1 domain, or (3) human and rabbit vWF A1 domains, using a nucleotide pool consisting of deoxy-nucleotides (DNA).
  • the selection strategy yielded high affinity aptamers specific for human and rabbit vWF A1 domains which had been immobilized on a hydrophobic plate.
  • a DNA template with the sequence 5′-CTACCTACGATCTGACTAGC NNNNNNGCTTACTCTCATGTAGTTCC-3′ (ARC 493) was synthesized using an ABI EXPEDITETM DNA synthesizer, and deprotected by standard methods.
  • the series of N's in the DNA template (SEQ ID NO 51) can be any combination of nucleotides and gives rise to the unique sequence region of the resulting aptamers.
  • the template was PCR amplified with the primers (5′-CTACCTACGATCTGACTAGC-3′) (SEQ ID NO 52) and (5′-AGGAACTACATGAGAGTAAGC(OH)-3′) (SEQ ID NO 53) under standard conditions.
  • the PCR product was subjected to alkaline hydrolysis (333 mM NaOH, 90° C., 15 min) followed by precipitation.
  • the strands were separated on a 10% denaturing polyacrylamide gel and the single stranded DNA pool, which migrated with a lower mobility, was excised from the gel, passively eluted, and precipitated with isopropanol.
  • each round was initiated by immobilizing 24 pmoles of rabbit vWF A1 (SEQ ID NO 6) domain under the same conditions.
  • 12 pmoles of human vWF A1 domain (SEQ ID NO 4) and 12 pmoles of rabbit vWF A1 domain (SEQ ID NO 6) were immobilized to a hydrophobic plate as previously described.
  • the protein target was alternated each round between human and rabbit vWF A1 domain, except in Round 11 human full length vWF (SEQ ID NO 7) was used.
  • the pool DNA bound to the immobilized protein target was eluted with 2 ⁇ 100 ⁇ L washes with elution buffer (preheated to 90° C., 7 M Urea, 100 mM NaOAc pH 5.3, 3 mM EDTA) for five minutes. Both elutions were pooled and precipitated by the addition of ethanol, then amplified in an initial PCR reaction (100 ⁇ L reactions including the 5′-primer according to SEQ ID NO 52, and the 3′-primer according to SEQ ID NO 53, and Taq polymerase (New England BioLabs, Cat. #M0267L, Beverly, Mass.). PCR reactions were done under the following conditions: a) denaturation step: 94° C.
  • PCR Threshold 10 ⁇ L of the PCR product was added to another 300 ⁇ L of PCR mix for a prep-scale PCR reaction.
  • the prep-scale PCR product was ethanol precipitated and was subjected to alkaline hydrolysis (333 mM NaOH, 90° C., 15 min).
  • the strands were separated on a 10% denaturing polyacrylamide gel and the single stranded DNA pool, which migrated with a lower mobility, was excised from the gel, passively eluted, and precipitated with isopropanol. In all cases, an equivalent of half of the total single stranded DNA product was carried forward as the starting pool for the subsequent round of selection.
  • the selection progress was monitored using a sandwich filter binding assay.
  • the 5′- 32 P-labeled pool DNA (trace concentration) was incubated with either a no target protein control, 100 nM human vWF A1 domain (SEQ ID NO 4), or 100 nM rabbit vWF A1 domain (SEQ ID NO 6), in 1 ⁇ Dulbecco's PBS containing 0.1 mg/mL tRNA, and 0.1 mg/mL salmon sperm DNA in a (final volume of 50 uL) for 30 minutes at room temperature and then applied to a nitrocellulose and nylon filter sandwich in a dot blot apparatus (Schleicher and Schuell, Keene, N.H.).
  • the percentage of pool DNA bound to the nitrocellulose was calculated after Rounds 6, 9, and 12 with a three point screen (no protein target control, 100 nM human vWF A1 domain (SEQ ID NO 5), 100 nM rabbit vWF A1 domain (SEQ ID NO 6). Pool binding was compared to that of the na ⁇ ve pool DNA (Round 0). The results of the DNA pool binding analyses are found in Table 13.
  • K D 's were determined for 10 of the vWF dependent binding sequences.
  • aptamers were 5′ end labeled with ⁇ - 32 P ATP and a competition dot blot assay was used with a constant protein concentration of 100 nM and an 8 point cold competitor DNA titration (333 nM, 100 nM, 33 nM, 10 nM, 3 nM, 1 nM, 33 pM, 0 pM) in 1 ⁇ DPBS plus 0.1 mg/mL BSA at room temperature for 30 minutes.
  • nucleic acid sequences of the DNA aptamers characterized in Table 14 above are described below, The unique sequence of each aptamer below begins at nucleotide 21, immediately following the sequence CTACCTACGATCTGACTAGC (SEQ ID NO 52), and runs until it meets the 3′ fixed nucleic acid sequence GCTTACTCTCATGTAGTTCC (SEQ ID NO 223).
  • a single set of DNA selections were done using full length human vWF and rabbit vWF domain A1 in a cross selection. While not wishing to be bound by any theory, our hypothesis is that such a selection should require successfully selected aptamers to bind to full length vWF, to bind to the A1 domain specifically and to cross react between human and rabbit proteins.
  • the dominant sequence family from this second set of DNA selections binds to full length human vWF, rabbit vWF domain A1 and is functional in both the FACS and BIPA biological assays as described in Example 3 below.
  • a DNA template with the sequence 5′-CTACCTACGATCTGACTAGC NNNNNGCTTACTCTCATGTAGTTCC-3′ (ARC 493) was synthesized using an ABI EXPEDITETM DNA synthesizer, and deprotected by standard methods.
  • the series of N's in the DNA template (SEQ ID NO 51) can be any combination of nucleotides and gives rise to the unique sequence region of the resulting aptamers.
  • the template was PCR amplified with the primers (5′-CTACCTACGATCTGACTAGC-3′) (SEQ ID NO 52) and (5′-AGGAACTACATGAGAGTAAGC(OH)-3′) (SEQ ID NO 53) under standard conditions.
  • the PCR product was subjected to alkaline hydrolysis (333 mM NaOH, 90° C., 15 min) followed by precipitation.
  • the strands were separated on a 10% denaturing polyacrylamide gel and the single stranded DNA pool, which migrated with a lower mobility, was excised from the gel, passively eluted, and precipitated with isopropanol.
  • the pool DNA was incubated for 1 hour at room temperature in an unblocked well to remove any plastic binding sequences from the pool.
  • the DNA was transferred to a BSA blocked well (not containing the protein target) for 1 hour at room temperature to remove any BSA binding sequences from the pool prior to the positive selection.
  • 0.1 mg/mL tRNA and 0.1 mg/mL salmon sperm DNA were spiked into the positive selection reaction as non-specific competitors.
  • the pool DNA bound to the immobilized protein target was eluted with 2 ⁇ 100 ⁇ L washes with elution buffer (preheated to 90° C., 7 M Urea, 100 mM NaOAc pH 5.3, 3 mM EDTA) for five minutes. Both elutions were pooled and precipitated by the addition of ethanol, then amplified in an initial PCR reaction (100 ⁇ L reactions including the 5′-primer according to SEQ ID NO 52, the 3′-primer according to SEQ ID NO 53, and Taq polymerase, (New England BioLabs, Cat. #M0267L, Beverly, Mass.). PCR reactions were done under the following conditions: a) denaturation step: 94° C.
  • PCR Threshold 10 ⁇ L of the PCR product was added to another 300 L of PCR mix for a prep-scale PCR reaction.
  • the prep-scale PCR product was ethanol precipitated and was subjected to alkaline hydrolysis (333 mM NaOH, 90° C., 15 min).
  • the strands were separated on a 10% denaturing polyacrylamide gel and the single stranded DNA pool, which migrated with a lower mobility, was excised from the gel, passively eluted, and precipitated with isopropanol. In all cases, an equivalent of half of the total single stranded DNA product was carried forward as the starting pool for the subsequent round of selection.
  • the selection progress was monitored using a sandwich filter binding assay.
  • the 5′- 32 P-labeled pool DNA (trace concentration) was incubated with either a no target protein control, 100 nM full length human vWF (Calbiochem Cat. #681300, La Jolla, Calif.), or 100 nM rabbit vWF A1 domain, in 1 ⁇ Dulbecco's PBS containing 0.1 mg/mL tRNA, and 0.1 mg/mL salmon sperm DNA, and 0.1 mg/mL BSA in a (final volume of 50 uL) for 30 minutes at room temperature and then applied to a nitrocellulose and nylon filter sandwich in a dot blot apparatus (Schleicher and Schuell, Keene, N.H.).
  • the percentage of pool DNA bound to the nitrocellulose was calculated after Rounds 7 and 9 by screening with a no protein target control, 30 nM/100 nM full length human vWF (Calbiochem Cat. #681300, La Jolla, Calif.), and 30 nM/100 nM rabbit vWF A1 domain (SEQ ID NO 6). Pool binding was compared to that of the na ⁇ ve pool DNA (Round 0). The results of the DNA pool binding analyses are found in Table 16 below.
  • the pools were cloned using the TOPO TA cloning kit (Invitrogen Cat. #45-0641, Carlsbad, Calif.) according to the manufacturer's instructions. Round 7 and 11 pool templates were cloned and sequenced (218 total sequences), producing 146 unique clones within 12 sequence families of which sequences from six families show vWF target binding activity. All unique clones were assayed twice in a 3-point dot blot screen (no protein target control, 20 nM full length human vWF (Calbiochem Cat.
  • K D s were determined for 3 of the vWF dependent binding sequences using the dot blot assay.
  • aptamers were 5′end labeled with ⁇ - 32 P ATP and were tested for direct binding to full length human vWF and rabbit vWF A1 domain.
  • a 12 point protein titration was used in the dot blot assay (300 nM, 100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 300 pM, 100 pM, 30 pM, 10 pM, 3 pM, 0 pM) in 1 ⁇ DPBS plus 0.1 mg/mL BSA at room temperature for 30 minutes.
  • nucleic acid sequences of the DNA aptamers characterized in Table 17 above are described below.
  • the unique sequence of each aptamer below begins at nucleotide 21, immediately following the sequence CTACCTACGATCTGACTAGC (SEQ ID NO 52), and runs until it meets the 3′ fixed nucleic acid sequence GCTTACTCTCATGTAGTTCC (SEQ ID NO 223).
  • the predicted secondary structure and core nucleic acid sequences required for binding to the vWF target of some embodiments of the invention comprised in Family #1 of this aptamer selection is depicted in FIG. 15 as SEQ ID NO 220.
  • aptamer ARC840 (AMX201.C8) (SEQ ID NO 23) was chosen from the rRfY selections for further characterization.
  • RNA transcript was labeled at the 5′-end with ⁇ - 32 P ATP and T4 polynucleotide kinase.
  • Radiolabeled ligands were subjected to partial alkaline hydrolysis and then selectively bound in solution to human von Willebrand Factor A1 domain (SEQ ID NO 5) at 500 nM before being passed through nitrocellulose filters. Both the retained and the not retained oligonucleotides were resolved separately on 8% denaturing polyacrylamide gels.
  • RNAstructure is a Windows implementation of the Zuker algorithm for RNA secondary structure prediction based on free energy minimization (Mathews, D. H.; Disney, M. D.; Childs, J. L.; Schroeder, S. J.; Zuker, M.; and Turner, D. H., “Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure,” 2004 . Proceedings of the National Academy of sciences, US, 101, 7287-7292). RNAstructure 4.1 uses the most current thermodynamic parameters from the Turner lab.
  • the purines comprise a 2′-OH and the pyrimidines comprise a 2′-F modification, while, the templates and primers comprise unmodified deoxyribonucleotides.
  • 5′-GGACCACCCUCGCAAGCAUUUUAAGAAUGACUUGUGCCGCUGGUCC-3′ 5′ PCR primer 5′-GATCGATCTAATACGACTCACTATA-3′ (SEQ ID NO 166) and 3′ PCR primer 5′-GGACCAGCGGCACAAGTC-3′ (SEQ ID NO 170) were used to amplify template 5′-GATCGATCTAATACGACTCACTATAGGACCACCCTCGCAAGCATTTTAAGAATGACT TGTGCCGCTGGTCC-3′ (SEQ ID NO171).
  • 5′-GGACCACCCUCGCAAGCAUUGAGAAAUGACUUGUGCCGCUGGUCC-3′ 5′ PCR primer 5′-GATCGATCTAATACGACTCACTATA-3′ (SEQ ID NO 166) and 3′ PCR primer 5′-GGACCAGCGGCACAAGTC-3′ (SEQ ID NO 170) were used to amplify template 5′-GATCGATCTAATACGACTCACTATAGGACCACCCTCGCAAGCATTGAGAAATGACTT GTGCCGCTGGTCC-3′ (SEQ ID NO 173).
  • 5′-GGACCACCCUCGCAACGAGAGUUGUGCCGCUGGUCC-3′ 5′ PCR primer 5′-GATCGATCTAATACGACTCACTATA-3′ (SEQ ID NO 166 ) and 3′ PCR primer 5′-GGACCAGCGGCACAACTC-3′ (SEQ ID NO 175) were used to amplify template 5′-GATCGATCTAATACGACTCACTATAGGACCACCCTCGCAACGAGAGTTGTGCCGCTG GTCC-3′ (SEQ ID NO 176).
  • RNA transcripts were labeled at the 5′-end with ⁇ - 32 P ATP and T4 polynucleotide kinase.
  • Radiolabeled ligands were subjected to partial alkaline hydrolysis and then selectively bound in solution to human vWF A1 domain (SEQ ID NO 5) at 500 nM before being passed through nitrocellulose filters. Retained oligonucleotides were resolved on 8% denaturing polyacrylamide gels. The smallest oligonucleotide bound to vWF defined the 3′-boundary.
  • the purines are 2′-OH purines and the pyrimidines are deoxy-pyrimidines, while the templates and primers comprise unmodified deoxyribonucleotides.
  • the following three minimized aptamer sequences were derived from DL.159.87.70 (SEQ ID NO 44):
  • aptamer sequence 5′-GGAGCCACGGGGTGGGTAGACGGCGGGTATGTGGCTCC-3′ (SEQ ID NO 180), 5′ PCR primer 5′-GATCGATCTAATACGACTCACTATA-3′ (SEQ ID NO 166) and 3′ PCR primer 5′-GGAGCCACATACCCGCCG-3′ (SEQ ID NO 181), were used to amplify template 5′-GATCGATCTAATACGACTCACTATAGGAGCCACGGGGTGGGTAGACGGCGGGTATG TGGCTCC-3′ (SEQ ID NO 182).
  • aptamer sequence 5′-GGAGCCACACGACATTGGCGCGAGAGCGCATGGCTCC-3′ (SEQ ID NO 201), 5′ PCR primer 5′-GATCGATCTAATACGACTCACTATA-3′ (SEQ ID NO 166) and 3′ PCR primer 5′-GGAGCCATGCGCTCTCG-3′ (SEQ ID NO 202), were used to amplify template 5′-GATCGATCTAATACGACTCACTATAGGAGCCACACGACATTGGCGCGAGAGCGCAT GGCTCC-3′ (SEQ ID NO 203).
  • SEQ ID NO 204 5′-CCAGCGGAATGAGAATGCTGATGGATTGCTCAGGTCTGCTGG -3′
  • ARC 845 SEQ ID NO 205
  • SEQ ID NO 206 5′-CGATCTGACTAGCGGAATGAGAATGCTGGTGGATCG -3′
  • SEQ ID NO 207 5′-GATCTGACTAGCGCAATGAGGATGCdTGATGGATTGCTCAGGTC - 3′
  • minimers were tested for binding to human vWF A1 domain (SEQ ID NO 5) using a competition dot blot assay with a constant protein concentration of 10 nM and a 12 point cold competitor DNA titration (3 uM, 1 uM, 333 nM, 100 nM, 33 nM, 10 nM, 3.3 nM, 1 nM, 333 pM, 100 pM, 33.3 pM, 0 pM) in 1 ⁇ Dulbecco's PBS containing 0.1 mg/mL BSA (final volume of 50 uL) for 30 minutes at room temperature.
  • ARC 845 (SEQ ID NO 205) represents the core nucleic acid binding sequence of the DNA SELEXTM 1, Family 1 aptamers.
  • nucleic acid sequences for above-described minimized DNA aptamers are as follows:
  • SEQ ID NO 208 SEQ ID NO 211
  • SEQ ID NO 208 demonstrated activity in the cellular assays (see Example 3, below).
  • ARC1027-1031 (SEQ ID NOS 212-216) further tested our hypothesis about the folding and secondary structure of aptamers AMX237.G6 (SEQ ID NO 114), AMX238.E9 (SEQ ID NO 115), and AMX238.H5 (SEQ ID NO 118) (see FIGS. 5 , 14 and 15 ).
  • the minimized sequences that showed potent activity in the cellular assays as described in Example 3 below were 5- 32 P end-labeled with ⁇ -32P ATP, and then desalted using two Centri-Spin 10 columns (Princeton Separations Cat. #CS-101, Adelphia, N.J.). Minimers were tested for direct binding to full length human vWF (SEQ ID NO 7), and rabbit vWF A1 domain (SEQ ID NO 6) using a 9 point protein titration (100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 300 pM, 100 pM, 30 pM, 0 pM) (see FIG.
  • ARC1029 Highly stable and potent variants of ARC1029 (SEQ ID NO 214) were identified through a systematic synthetic modification approach involving 5 phases of aptamer synthesis, purification and assay for binding activity.
  • the PEG spacer of ARC1029 (SEQ ID NO 214 was replaced with a short oligonucleotide sequence, dTdTdC, resulting in ARC1115 (SEQ ID NO 221) as seen in FIG. 16 and Table 21 below.
  • a highly stabilizing 3′-inverted dT was synthesized on the three prime end of ARC1115 (SEQ ID NO 221) resulting in ARC1172 (SEQ ID NO 222) (SEQ ID NO 222) also as seen in FIG.
  • each individual residue in ARC1172 was replaced by the corresponding 2′-O methyl containing residue (with dT being replaced by mU unless otherwise specified) resulting in ARC1194 (SEQ ID NO 223)-ARC1234 as shown in Table 21 below and FIG. 16 .
  • ARC1194 SEQ ID NO 223-ARC1234 as shown in Table 21 below and FIG. 16 .
  • a set of composite replacements were made in the stem regions of ARC1172 (SEQ ID NO 222) resulting in ARC1235 to 1243 also as shown in Table 21 and in FIG. 16 .
  • ARC1338 SEQ ID NO 273 to 1342 were synthesized with block modifications based on the tolerated individual substitutions from phase 1 modification.
  • ARC1343 SEQ ID NO 278)-ARC1345 were synthesized each with a different phosphorothioate phosphate backbone modification (see FIG. 17 and Table 22 below)
  • ARC1361 (SEQ ID NO 284) to ARC1381 (SEQ ID NO 304), shown in Table 23 and FIG. 17 were synthesized during phase 3 of the aptamer modification process.
  • ARC1346 (SEQ ID NO 281) was synthesized with a mC to dC modification at position 36 resulting in ARC1361 (SEQ ID NO 284) as shown in Table 23 below.
  • ARC1361 (SEQ ID NO 284) served as the base sequence for introduction of single phosphorothioate phosphate backbone modifications that resulted in ARC1362 to ARC1381 (SEQ ID NO 304) also shown in Table 23 below.
  • ARC1368 SEQ ID NO 291
  • ARC1172 SEQ ID NO 222
  • ARC1524 SEQ ID NO 305
  • ARC1535 SEQ ID NO 316
  • ARC1546 SEQ ID NO 3107
  • ARC1759 SEQ ID NO 3148
  • ARC1759 which is identical to ARC1172 (SEQ ID NO 222) except that it has single phosphorothioate substitution between the G at position 21 and the T at position 22 showed measurable improvement in affinity relative to ARC1172 (SEQ ID NO 222).
  • Polyethylene glycol moieties were conjugated to the 5′ terminus of ARC1368 (SEQ ID NO 291) and ARC1172 (SEQ ID NO 222) via amine reactive chemistries.
  • the NH2-mGmCmGmUdGdCdAmGmUmGmCmCmUmUmCmGmGmCdCmG-s-dTmGdCdGdGdTmGmCdCmUdCdCmGmUdCmAmCmGmC-3T (ARC1368 (SEQ ID NO 291) with a 5′ hexylamine modification) and ARC1884 (SEQ ID NO 322) NH2-dGdGdCdGdTdGdCdAdGdTdGdCdCTdTdCdGdGdCdCdGTdGdCdGdGTdGdCdCTdCdCdGT dCdAdCdGdCdCdCdC-3T
  • the amine-modified aptamers were conjugated to different PEG moieties, as indicated in Table 25 below, post-synthetically.
  • botrocetin In one assay botrocetin is used.
  • Botrocetin a protein isolated from snake venom, is known to induce von Willebrand Factor binding to the gpib receptor on live and fixed platelets. This reaction causes agglutination of suspensions of fixed platelets via vWF multimerization.
  • PRP platelet rich plasma
  • vWF/botrocetin induction of agglutination is followed by a second phase of platelet aggregation caused by metabolic activation of the platelets.
  • the amount of vWF bound to fixed platelets can be measured with an antibody to vWF.
  • the fluorescence signal from bound antibody incubated with a fluorescein conjugated secondary antibody is then detected and quantified by flow cytometry.
  • the ability of an aptamer of the invention to block vWF binding to platelets is correlated with a reduction in fluorescence signal.
  • Botrocetin induces the binding of the A1 domain of vWF to platelets, as well as the full length protein. It was determined by the inventors that 6-Histidine-tagged rabbit A1 domain vWF purified protein could be induced to bind to human lyophilized platelets with botrocetin. Rabbit A1 binding to platelets is measured with an anti-poly-His antibody followed by incubation with a phycoerythrin conjugated secondary antibody. The degree of binding can be quantified by flow cytometric analysis. The ability of aptamers to block the binding of rabbit A1 to human fixed platelets was correlated with decreased fluorescence signal.
  • botrocetin induces platelet aggregation via vWF.
  • Platelet aggregate formation can be measured optically as an increase in % light transmittance on a Chronolog Model 490-4D Aggregometer because aggregation of platelets clarifies the plasma.
  • Aptamers of the invention were analyzed for their ability to inhibit botrocetin induced platelet aggregation (“BIPA”) in human blood.
  • BIPA botrocetin induced platelet aggregation
  • An aptamer of the invention was considered to be active if it could prevent aggregate formation for six minutes post botrocetin addition.
  • Another assay of this Example, using the PFA-100 instrument is an agonist independent but vWF dependent assay that uses the PFA-100 instrument (Harrison et al., Clin. Lab. Haem., v24, p 225-32 (2002)).
  • the PFA-100 simulates the formation of a hemostatic plug under conditions of high shear force in vivo by recording the time required for a platelets to aggregate and block the flow of citrated whole blood through a microscopic aperture in a membrane coated with collagen and either epinephrine or ADP.
  • This activity is von Willebrand factor dependent as high MW vWF multimers bind to immobilized collagen on the membrane and then bind to and activate platelets because of the shear force induced by drawing the blood through the microscopic aperture.
  • this assay is complimentary to the BIPA and FACS assays in that it is vWF dependent, however it has some advantages in that it does not require the addition of the vWF agonist botrocetin and uses whole blood instead of platelet rich plasma.
  • FIG. 1 Another assay of this Example used ADP to induce platelet aggregation.
  • Aggregation of platelet rich plasma (PRP) can be in induced in multiple ways.
  • the snake venom protein botrocetin acts on vWF as described above, stabilizing its interaction with the platelet receptor gpIb thereby inducing platelet aggregation.
  • Binding of vWF to gpIb is an early step in platelet aggregation, thus there is an expectation that inhibitors that block downstream components of the aggregation process (i.e., the IIbIIIa antagonists IntegrelinTM and ReoProTM) would also prevent botrocetin induced platelet aggregation.
  • vWF antagonists In the case of agonists that act directly on platelets and induce aggregation (ADP for example), one would expect that antagonists upstream of the agonist would be ineffective (an anti-vWF aptamer for example), while antagonists that act directly on platelets (IIbIIIa antagonists) would remain potent.
  • the specificity of a vWF antagonist relative to a IIbIIIa antagonist will increase the safety of the anti-vWF antagonist by decreasing the bleeding time associated with treatment.
  • platelet aggregation occurs as platelets bind to collagen immobilized vWF on the surface of the plaque.
  • vWF/gpIb interaction both inhibiting the vWF/gpIb interaction as well as blocking the IIbIIIa receptor binding to fibrin will prevent platelet aggregation.
  • the biological specificity conferred by targeting vWF insures that unlike anti-IIbIIIa treatment, platelets themselves are not targeted directly insuring they can still be activated by other means, thus reducing potential bleeding complications associated with anti-platelet therapy.
  • vWF von Willebrand Factor
  • Bovine serum albumin purchased from Calbiochem (Cat#681300 and #126593, respectively) (La Jolla, Calif.); domain A1 rabbit vWF (SEQ ID NO 6) was expressed and purified using standard methods and conditions. Lyophilized human platelets (P/N 299-2), cuvettes (P/N 312), stir bars (P/N 311), platelet aggregometer (model 490-4D), and AGGRO/LINK Software were purchased from Chronolog (Haverton, Pa.). Botrocetin (12201-100U-B) was manufactured by Pentapharm (Basel, Switzerland).
  • Fresh blood was obtained from apparently healthy, nonsteroidal anti-inflammatory drug (“NSAID”) free donors and was drawn into 5 mL 0.105 M Sodium Citrate Vacutainer tubes (Cat#369714) (Becton Dickinson—Franklin Lakes, N.J.).
  • Physiological saline was manufactured by Aldon (Cat #9420306) (Avon, N.Y.) and phosphate buffered saline (Cat #21-040-CV) was purchased from Cellgro (Herndon, Va.).
  • Flow cytometric experiments were performed on a BD Biosciences FACSCAN machine and analyzed with CellQuest Software (San Jose, Calif.).
  • Anti-von Willebrand Factor mouse monoclonal antibody (Cat#GTI-V1A) was purchased from GTI (Waukesha, Wis.). Penta-HIS-biotin conjugate monoclonal antibody (Cat #34440) was purchased from Qiagen (Germany). Anti-mouse IgG2a—FITC conjugate (Cat #553390) was purchased from BD Biosciences (San Diego, Calif.). Anti-mouse IgG-PE conjugate antibody (Cat #715-116-150) was purchased from Jackson ImmunoResearch Laboratories (West Grove, Pa.).
  • aptamers potency to block human vWF binding to lyophilized platelets was assessed by flow cytometric analysis. Titrations of aptamers (0 nM, 0.1 nM to 1000 nM) were pre-incubated briefly with 5 nM of full length human vWF in FACS buffer (PBS plus 0.5% bovine serum albumin) at room temperature in a volume of 50 uL. Another 50 uL containing 5 uL of lyophilized platelets plus 1 uL of 0.1 U/uL of botrocetin in FACS buffer was added to aptamer/vWF. This reaction was allowed to proceed for 15 minutes at 37 degrees C. after which 200 uL of FACS buffer was added.
  • FACS buffer PBS plus 0.5% bovine serum albumin
  • Platelets were collected by a 6 minute spin at 1470 RCF and the supernatant was discarded.
  • the pellets were resuspended in 100 uL of FACS buffer containing a 1:100 dilution of anti-vWF antibody and were incubated at room temperature for 30 minutes. After dilution with 200 uL of FACS buffer, the platelets were spun at 1470 RCF for 6 minutes and the supernatant was discarded.
  • the pellets were resuspended in a 1:100 solution of anti-IgG2a-FITC antibody and were incubated in the dark for 30 minutes at room temperature.
  • the entire 100 uL was diluted into 200 uL of FACS buffer and analyzed immediately by flow cytometric analysis in the FACSCAN.
  • aptamers of the invention were also assessed by flow cytometric analysis. Titrations of aptamers (zero, 0.1 nM to 1000 nM) were preincubated briefly with 4 nM of rabbit A1 vWF in FACS buffer (PBS plus 0.5% bovine serum albumin) at room temperature in a volume of 50 uL. Another 50 uL containing 5 uL of lyophilized platelets plus 1 uL of 0.1 U/uL of botrocetin in FACS buffer was added to aptamer/vWF. This reaction was allowed to proceed for 15 minutes at 37 degrees C. after which 200 uL of FACS buffer was added.
  • FACS buffer PBS plus 0.5% bovine serum albumin
  • Platelets were collected by a 6 minute spin at 1470 RCF and the supernatant was discarded.
  • the pellets were resuspended in 100 uL of FACS buffer containing a 1:200 dilution of anti-Penta-HIS-biotin conjugate antibody and were incubated at room temperature for 30 minutes. After the dilution with 200 uL of FACS buffer, the platelets were spun at 1470 RCF for 6 minutes, and the supernatant was discarded.
  • the pellets were resuspended in a 1:100 solution of anti-IgG-PE antibody and were incubated in the dark for 30 minutes at room temperature.
  • BIPA assays were done using freshly prepared platelet rich plasma. Blood was obtained from healthy human donors who had not taken NSAIDS for at least five days. 213 ⁇ 4 gauge butterfly needles (Cat# 367287) from Becton Dickinson were used to draw blood into 0.105 M sodium citrate vacutainer tubes. Collected blood was pooled into 15 mL conical tubes and was spun at 200 g for 20 minutes. The turbid, yellow layer of platelet rich plasma (“PRP”) was withdrawn from the tubes, pooled, and set aside at room temperature. The remaining blood was spun at 2500 g for ten minutes.
  • PRP turbid, yellow layer of platelet rich plasma
  • PRP platelet poor plasma
  • the clarified layer of plasma was withdrawn and set aside at room temperature.
  • PRP was aliquoted into cuvettes containing stir bars at a volume of 470 uL.
  • a sample of 500 uL of PPP was aliquoted into a cuvette and placed in the PPP reference cell of the platelet aggregometer.
  • Samples of PRP were prewarmed at 37 degrees C. in the platelet aggregometer for 3-5 minutes before being used in BIPA assays.
  • the concentration of botrocetin needed to induce platelet aggregation for each individual donor was determined by titration. This concentration of botrocetin was used for the remainder of the experiment.
  • aptamer was assayed by adding titrations of aptamer (zero, 1 nM to 1000 nM) to prewarmed PRP for one minute, followed by addition of botrocetin. With platelet aggregation, an increase in amplitude of light transmission is seen ( FIG. 8 ). The concentration at which platelet aggregation is blocked at 90% or greater after 6 minutes is reported in the final column of Table 26.
  • AUC area under the curve
  • ARC1172 (SEQ ID NO 222), ARC1346 (SEQ ID NO 281), ARC1368 (SEQ ID NO 291), ARC1525 (negative control), ARC1779 (SEQ ID NO 320), ARC1780 (SEQ ID NO 321), and ARC1885 (SEQ ID NO 323) identified in the medchem modification process described in Example 2 above, were tested in a series of biological assays. These assays included the FACS (as described in Example 3A and 3B) and BIPA assays (as described in Example 3C), as well as the platelet PFA-100 assay described below.
  • NSAID non-steroidal anti-inflammatory drug
  • a self-test which included an O-ring cleaning process, was always run on the PFA-100 machine before running any samples to ensure proper function of the machine.
  • Fresh whole blood was collected from healthy donors or cynomolgus macaques, as indicated in Table 27 below, who/that had not taken NSAIDs for at least three days.
  • Blood from human donors was collected into 5 ml 0.105M sodium citrate tubes using a 213 ⁇ 4 gauge butterfly needle, and the tubes were gently inverted three times to ensure mixture of blood with sodium citrate. During the entire experiment, the tubes of whole blood were gently inverted every five minutes to prevent settling.
  • the aptamer was added to no-additive vacutainer tubes and diluted to the desired concentrations (ex: 0 nM, 1 nM to 1000 nM) using physiological saline such that the final volume was 601.
  • the desired concentrations ex: 0 nM, 1 nM to 1000 nM
  • 1940 ⁇ l of whole blood was added to the tube containing a concentration of aptamer. This tube was gently inverted three times to thoroughly mix the aptamer and blood. Samples were always run in duplicate on the PFA-100 machine. 800 ⁇ l of this blood mixture was loaded into the collagen/epinephrine test cartridges. The test cartridges were loaded onto the PFA-100 machine. The time of occlusion of the aperture was measured by the PFA-100, with a maximal time of 300 seconds. We estimate the IC 95 in this assay to be the minimum concentration of aptamer that extends the closing time to 300 seconds.
  • FIG. 20 depicts clotting time in human whole blood as a function of aptamer concentration in the PFA-100 assay for ARC1368 (SEQ ID NO 291) and the negative control ARC1525. Additional results of the FACS, BIPA and PFA-100 assays are tabulated in Table 27 below.
  • ARC1346 50 180 >1000 ND ND ARC1368 2 4.0 ⁇ 200 ⁇ 100 ⁇ 100 ARC1525 ND ND no no ND inhibition inhibition ARC1779 ND ND ⁇ 100 ⁇ 100 ND ARC1780 ND ND ⁇ 100 ⁇ 100 ND ARC1885 ND ND ⁇ 50 ⁇ 100 ND
  • ARC1368 SEQ ID NO 291
  • IntegrillinTM IntegrillinTM
  • ReoPrOTM ADP Induced Platelet Aggregation assays.
  • AIPA was performed with human PRP exactly as was done for BIPA described above in Example 3C with the exception that instead of adding botrocetin, 10 micromolar ADP (Chronolog, Haverton, Pa.) was added to induce platelet aggregation.
  • the PFA-100 results are shown in FIG. 21 .
  • the BIPA results are shown in FIG. 22 .
  • the AIPA results are shown in FIG. 23 . As can be seen in FIGS.
  • ARC1368 shows potency comparable to ReoPrOTM in PFA-100 and BIPA assays. Consistent with the vWF dependent mechanism described above, the anti-vWF aptamer shows no ability to block AIPA while the IIbIIIa antagonists remain potent in that assay as shown in FIG. 23 .
  • ARC1172 SEQ ID NO 222
  • ARC1346 SEQ ID NO 281
  • ARC1368 SEQ ID NO 291
  • ARC1533 was assayed for nuclease stability in both human and rat plasma.
  • Plasma nuclease degradation was measured on denaturing polyacrylamide gel electrophoresis as described below. Briefly, for plasma stability determination, chemically synthesized aptamers were purified using denaturing polyacrylamide gel electrophoresis, 5′ end labeled with ⁇ - 32 P ATP and then gel purified again. Trace 32-P labeled aptamer was incubated in the presence of 100 nM unlabeled aptamer in 95% human or rat plasma in a 200 microliter binding reaction.
  • reaction for the time zero point was made separately with the same components except that the plasma was replaced with PBS. This insured that the amount or radioactivity loaded on gels was consistent across an experiment.
  • Reactions were incubated at 37° C. in a thermocycler for the 1, 3, 10, 30 and 100 hours unless otherwise specified. At each time point, 20 microliters of the reaction was removed, combined with 200 microliters of formamide loading dye and flash frozen in liquid nitrogen and stored at ⁇ 20° C. After the last time point was taken, frozen samples were thawed and 20 microliters was removed from each time point. SDS was then added to the small samples to a final concentration of 0.1%. The samples were then incubated at 90° C.
  • T 1/2 The half-life of the aptamer (T 1/2 ) is equal to the (ln 2)/m2.
  • ARC1368 SEQ ID NO 291
  • 1779 SEQ ID NO 320
  • 1780 SEQ ID NO 321
  • ARC1368 SEQ ID NO 291
  • 1779 SEQ ID NO 320
  • 1780 SEQ ID NO 321
  • BIPA Botracetin-induced platelet aggregation
  • the primate plasma aptamer concentration plotted as a function of time is depicted in the graph of FIG. 25 .
  • the mean concentration-times profiles based on the OliGreenTM assay showed that the pharmacokinetic profiles of ARC1368 (SEQ ID NO 291), ARC1780 (SEQ ID NO 321) and ARC1779 (SEQ ID NO 320) were mainly monophasic.
  • the unPEGylated aptamer (ARC1368 (SEQ ID NO 291)) displayed a rapid distribution phase compared to ARC1779 (SEQ ID NO 320) and ARC1780 (SEQ ID NO 321).
  • ARC1780 Unlike ARC1368 (SEQ ID NO 291), the 40 kDa PEG conjugate ARC1780 (SEQ ID NO 321) displayed prolonged distribution phase compared to ARC1779 (SEQ ID NO 320). ARC1779 (20 kDa PEG) displayed a distribution phase with ⁇ -half-life of ⁇ 2 hr.
  • ARC1779 -PK/PD correlation of ARC1779 was evaluated in three cynomolgus macaques after a single intravenous (IV) bolus dose at 0.5 mg/kg.
  • IV intravenous
  • ARC1779 levels in the plasma were correlated to PD effects of ARC1779 in inhibition of platelet function or prolongation of cutaneous bleeding time (CBT).
  • PK and PD analysis were performed percutaneously at various time points post-dose for PK and PD analysis.
  • PD effect of ARC1779 on platelet function was determined by PFA-100 assays and effect of ARC1779 on bleeding was measured by CBT time.
  • plasma samples were analyzed by HPLC for quantitation of ARC1779 levels.
  • PK parameter estimates were determined by 2-compartment analysis. The results are presented in FIG. 26 .
  • the concentration-time profiles generated for individual monkeys showed predominantly the distribution phase of ARC1779 pharmacokinetics
  • the distribution half-life was determined to be approximately 1.0 hour (t 1/2 ⁇ ).
  • the elimination half-life (t 1/2 ⁇ ), was not well-determined from the available data.
  • FIG. 26 The PD effect of ARC1779 on platelet aggregation measured by the PFA-100 assay is shown in FIG. 26 .
  • the plasma concentrations of ARC1779 were in excess of 300 nM, platelet function was inhibited as assessed by the PFA-100 instrument.
  • plasma aptamer concentrations decreased to approximately 77 nM, platelet function returned to normal.
  • ARC1779 inhibited platelet function in vivo at a plasma concentration of approximately 300 nM. This in vivo concentration was approximately 3-fold higher than the observed concentration of aptamer necessary to inhibit platelet function in vitro. In contrast, even at high plasma concentrations (1000 nM), ARC1779 showed minimal effect on cutaneous bleeding time following single bolus dosing.
  • a blood pressure cuff was applied to the biceps region of the forearm to be tested and inflated to maintain a constant pressure of 40 mmHg.
  • a Surgicutt® Automated Incision Device ITC, Edison, N.J.
  • a longitudinal incision was made over the lateral aspect of the volar surface of the forearm distal to the antecubital crease.
  • a stopwatch was started at the time of incision.
  • the blood was wicked with Surgicutt® Bleeding Time Blotting Paper (ITC, Edison, N.J.) while avoiding direct contact with the incision. Every 15-30 seconds the blotting paper was rotated and re-blotted at a fresh site on the paper. The blotting was repeated until blood was no longer wicked onto the paper or for 30 minutes whichever came first. Bleeding time is determined to within 30 seconds of the time when blood is no longer wicked onto the paper.
  • FIG. 27 shows the cutaneous bleeding time in minutes, BIPA IC 90 in nM and PFA IC 95 in nM at various time points, shown in column 1, relative to ARC1779 (SEQ ID NO 320) dosing for three different animals.
  • FIG. 28 shows a graph of the average PFA-100 closure time from the blood of three ARC1779 (SEQ ID NO 320) treated macaques taken at various time points following dosing.
  • FIG. 29 shows the cutaneous bleeding time of the three ARC1779 (SEQ ID NO 320) treated macaques taken at various time points following dosing.
  • FIG. 30 correlates the average cutaneous bleeding time in ARC1779 (SEQ ID NO 320) treated C.
  • FIGS. 27 to 30 at time points up to and including 2 hours, where BIPA and PFA-100 closure time were maximally inhibited, there is very little increase in cutaneous bleeding times.
  • concentrations of the anti-gpIIbIIIa antagonist IntegrilinTM that yield similar inhibition of platelet aggregation ex vivo, template or cutaneous bleeding times are between 20 and 30 minutes. See, e.g. Phillips, D. R. and Scarborough, R. M., Am J Cardiol, 80(4A): 11B-20B (1997).
  • Each animal was anesthetized prior to surgical preparation, intubated and maintained in anesthesia with isoflurane inhalant anesthetic to effect delivered through a volume-regulated respirator.
  • An intravenous catheter was also placed in a peripheral vein for administration of lactated Ringer's solution during the procedure.
  • a catheter was placed in the femoral artery of each animal for continuous monitoring of arterial blood pressure. Similarly a catheter was placed in the femoral vein for blood sample collection.
  • Each carotid artery was instrumented with a Doppler flow probe connected to a flow meter. The flow probes were placed around the artery at a point proximal to the insertion of the intra-arterial electrode and stenosis. The stenosis was placed around each carotid artery so that the blood flow was reduced by approximately 50% to 60% without altering the mean blood flow. Blood flow in the carotid arteries was monitored and recorded continuously throughout the observation periods.
  • Electrolytic injury to the intimal surface of each carotid artery was accomplished via placement of an intravascular electrode.
  • Each electrode was connected to the positive pole of a constant current device and cathode connected to a distant subcutaneous site. Continuous current was delivered to each vessel for a period of 3 hours or for 30 minutes after complete occlusion, whichever was shorter.
  • the animal was administered the test article as indicated in Table 32 above. Approximately 15 minutes after test article administration, the electrical current was applied at 100 ⁇ A. Blood samples and CBT measurements were collected at the time points specified in Blood Sample Collection Schedule as indicated in FIG. 31 . The current was terminated ⁇ 30 minutes after the blood flow signal remained stable at zero flow (which indicates an occlusive thrombus had been formed at the site) or after 180 minutes of electrical stimulation. Approximately 195 minutes after the test article was administered, the left carotid artery (“LCA”) had electrical current administered in a similar fashion as previously described for the RCA. After termination of all surgical procedures and sample collection each animal was euthanized.
  • LCA left carotid artery
  • the ARC1779 (SEQ ID NO 320) plasma concentration (as measured by HPLC) over time for each animal in treatment group 3 is depicted in FIG. 32 .
  • the time to occlusion measured via Doppler flow for each treatment group is indicated in FIG. 33 .
  • ARC1779 (SEQ ID NO 320) inhibited thrombus formation during sequential 180-minute electrical injuries to the carotid arteries of the cynomolgus monkeys in this animal thrombosis model.
  • Example 5B The study described in Example 5B above was extended to test the efficacy of ARC1779 (SEQ ID NO 320) in inhibiting intra-arterial thrombosis in the non-human primate electrolytic thrombosis model at lower aptamer dosage levels.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Surgery (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
US12/322,078 2004-09-07 2009-01-29 Aptamers to von Willebrand factor and their use as thrombotic disease therapeutics Expired - Fee Related US7998940B2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US12/322,078 US7998940B2 (en) 2004-09-07 2009-01-29 Aptamers to von Willebrand factor and their use as thrombotic disease therapeutics

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US60804704P 2004-09-07 2004-09-07
US66195005P 2005-03-11 2005-03-11
US67842705P 2005-05-06 2005-05-06
US69023105P 2005-06-13 2005-06-13
US11/222,346 US7566701B2 (en) 2004-09-07 2005-09-07 Aptamers to von Willebrand Factor and their use as thrombotic disease therapeutics
US12/322,078 US7998940B2 (en) 2004-09-07 2009-01-29 Aptamers to von Willebrand factor and their use as thrombotic disease therapeutics

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US11/222,346 Continuation US7566701B2 (en) 2004-09-07 2005-09-07 Aptamers to von Willebrand Factor and their use as thrombotic disease therapeutics

Publications (2)

Publication Number Publication Date
US20090149643A1 US20090149643A1 (en) 2009-06-11
US7998940B2 true US7998940B2 (en) 2011-08-16

Family

ID=36090463

Family Applications (1)

Application Number Title Priority Date Filing Date
US12/322,078 Expired - Fee Related US7998940B2 (en) 2004-09-07 2009-01-29 Aptamers to von Willebrand factor and their use as thrombotic disease therapeutics

Country Status (11)

Country Link
US (1) US7998940B2 (es)
EP (1) EP1789096A4 (es)
JP (1) JP2008512098A (es)
KR (1) KR20070101227A (es)
AU (1) AU2005287273B2 (es)
BR (1) BRPI0514984A (es)
CA (1) CA2579374A1 (es)
IL (1) IL181689A0 (es)
MX (1) MX2007002772A (es)
SG (1) SG156618A1 (es)
WO (1) WO2006033854A2 (es)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015117201A1 (en) * 2014-02-05 2015-08-13 Deakin University Aptamer construct
US10577610B2 (en) 2015-02-11 2020-03-03 Deakin University EpCAM aptamers and conjugates thereof
US10695362B2 (en) 2010-05-14 2020-06-30 Tagcyx Biotechnologies Inc. Stabilization method of functional nucleic acid

Families Citing this family (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1330544A4 (en) 2000-09-26 2005-04-06 Univ Duke RNA APTAMERS AND METHOD OF IDENTIFYING SAID APTAMERS
WO2005106042A2 (en) 2004-04-22 2005-11-10 Regado Biosciences, Inc. Improved modulators of coagulation factors
RU2008103346A (ru) * 2005-06-30 2009-08-10 Аркемикс Корп. (Us) Материалы и способы для получения полностью 2-модифицированных транскриптов нуклеиновых кислот
WO2008078180A2 (en) * 2006-12-22 2008-07-03 Archemix Corp. Materials and methods for the generation of transcripts comprising modified nucleotides
JP5349323B2 (ja) * 2007-01-10 2013-11-20 アーケミックス コーポレイション 修飾ヌクレオチドを含む転写物を生成させるための材料および方法
WO2008150495A2 (en) * 2007-06-01 2008-12-11 Archemix Corp. Vwf aptamer formulations and methods for use
RU2571660C2 (ru) * 2009-06-03 2015-12-20 ОБЩЕСТВО С ОГРАНИЧЕННОЙ ОТВЕТСТВЕННОСТЬЮ "НоваМедика" Модуляторы гликопротеина vi, представляющие собой нуклеиновую кислоту
JP2011024499A (ja) * 2009-07-27 2011-02-10 Nec Soft Ltd 核酸の製造方法、デリバリー対象物質を臓器特異的にデリバリー可能なデリバリー用タグ核酸およびその用途
US8236570B2 (en) 2009-11-03 2012-08-07 Infoscitex Methods for identifying nucleic acid ligands
US8841429B2 (en) 2009-11-03 2014-09-23 Vivonics, Inc. Nucleic acid ligands against infectious prions
JP5337096B2 (ja) * 2010-04-28 2013-11-06 株式会社日立製作所 動脈硬化の評価法
KR101250557B1 (ko) * 2011-05-18 2013-04-03 국립암센터 Ｐａｕｆ 특이적 앱타머 및 이를 포함하는 췌장암 치료용 조성물
CA2850198A1 (en) 2011-09-30 2013-04-04 Mallinckrodt Llc Remote assembly of targeted nanoparticles using complementary oligonucleotide linkers
CN104661664B (zh) 2012-07-13 2020-07-03 波涛生命科学有限公司 手性控制
JP6544750B2 (ja) * 2015-03-06 2019-07-17 タグシクス・バイオ株式会社 Ｄｎａアプタマーの安定化法
SG11201803631YA (en) * 2015-10-30 2018-05-30 Tagcyx Biotechnologies DNA aptamer that binds to vWF
WO2018053427A1 (en) 2016-09-16 2018-03-22 Duke University Von willebrand factor (vwf)-targeting agents and methods of using the same
WO2018213697A1 (en) * 2017-05-19 2018-11-22 Band Therapeutics, Llc Compositions and methods for the treatment of complications and disorders relating to von willebrand factor
KR20220084272A (ko) * 2019-07-26 2022-06-21 익사카 프랑스 세포 표적화 및 라벨링에 사용하기 위한 항-cd3 압타머
US20230038761A1 (en) * 2020-02-04 2023-02-09 Band Therapeutics, Llc Regulation of von willebrand factor (vwf)

Citations (112)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3710795A (en) 1970-09-29 1973-01-16 Alza Corp Drug-delivery device with stretched, rate-controlling membrane
US4666884A (en) 1984-04-10 1987-05-19 New England Deaconess Hospital Method of inhibiting binding of von Willebrand factor to human platelets and inducing interaction of platelets with vessel walls
GB2183661A (en) 1985-03-30 1987-06-10 Marc Ballivet Method for obtaining dna, rna, peptides, polypeptides or proteins by means of a dna recombinant technique
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4935363A (en) 1987-03-30 1990-06-19 Board Of Regents, The University Of Texas System Sterol regulatory elements
US4959309A (en) 1983-07-14 1990-09-25 Molecular Diagnostics, Inc. Fast photochemical method of labelling nucleic acids for detection purposes in hybridization assays
US5043429A (en) 1987-05-01 1991-08-27 Scripps Clinic And Research Foundation Protein fragments containing Factor VIII binding domain of von Willebrand Factor
US5070010A (en) 1989-10-30 1991-12-03 Hoffman-La Roche Inc. Method for determining anti-viral transactivating activity
US5112950A (en) 1987-08-11 1992-05-12 Transgene S.A. Factor viii analog, preparation process, and pharmaceutical composition containing it
US5171844A (en) 1987-06-12 1992-12-15 Gist-Brocades N.W. Proteins with factor viii activity: process for their preparation using genetically-engineered cells and pharmaceutical compositions containing them
US5180583A (en) 1985-11-26 1993-01-19 Hedner Ulla K E Method for the treatment of bleeding disorders
EP0197592B1 (en) 1985-04-01 1993-02-17 Stichting Vrienden Van De Stichting Dr. Karl Landsteiner Preparation of the human von willebrand factor by recombinant dna
US5200510A (en) 1987-06-16 1993-04-06 Zymogenetics, Inc. Method for purifying factor viii:c, von willebrand factor and complexes thereof
US5238919A (en) 1986-05-30 1993-08-24 Scipps Clinic And Research Foundation Peptides that inhibit von Willebrand Factor binding to the platelet SPIB receptor
US5262564A (en) 1992-10-30 1993-11-16 Octamer, Inc. Sulfinic acid adducts of organo nitroso compounds useful as retroviral inactivating agents anti-retroviral agents and anti-tumor agents
US5270163A (en) 1990-06-11 1993-12-14 University Research Corporation Methods for identifying nucleic acid ligands
US5298239A (en) 1991-10-07 1994-03-29 The Research Foundation Of State University Of New York Mutations rendering platelet glycoprotein IB α less reactive
US5317097A (en) 1991-10-07 1994-05-31 The Research Foundation Of State University Of New York Mutations in the gene encoding the α chain on platelet glycoprotein IB
US5318899A (en) 1989-06-16 1994-06-07 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5321127A (en) 1991-03-18 1994-06-14 Brigham And Women's Hospital Antiplatelet and antithrombotic activity of platelet glycoprotein Ib receptor fragments
US5336667A (en) 1991-12-03 1994-08-09 Temple University Of The Commonwealth System Of Higher Education Method for inhibiting the ahesion of platelet with alboaggregins: platelet agonists which bind to platelet membrane glycoprotein IB
US5338671A (en) 1992-10-07 1994-08-16 Eastman Kodak Company DNA amplification with thermostable DNA polymerase and polymerase inhibiting antibody
US5340727A (en) 1987-11-17 1994-08-23 The Scripps Research Institute GPIbα fragments and recombinant DNA expression vectors
US5364771A (en) 1992-04-07 1994-11-15 Emory University Hybrid human/porcine factor VIII
US5366869A (en) 1991-11-08 1994-11-22 Sheldon Goldstein Multiple coagulation test device and method
US5459015A (en) 1990-06-11 1995-10-17 Nexstar Pharmaceuticals, Inc. High-affinity RNA ligands of basic fibroblast growth factor
US5460950A (en) 1990-11-26 1995-10-24 Genetics Institute, Inc. Expression of PACE in host cells and methods of use thereof
US5462752A (en) 1994-07-28 1995-10-31 Prp, Inc. Inhibition of platelet binding
US5475096A (en) 1990-06-11 1995-12-12 University Research Corporation Nucleic acid ligands
US5496938A (en) 1990-06-11 1996-03-05 Nexstar Pharmaceuticals, Inc. Nucleic acid ligands to HIV-RT and HIV-1 rev
US5503978A (en) 1990-06-11 1996-04-02 University Research Corporation Method for identification of high affinity DNA ligands of HIV-1 reverse transcriptase
US5532255A (en) 1993-05-01 1996-07-02 Merck Patent Gesellschaft Mit Beschrankter Haftung Adhesion receptor antagonists
US5539086A (en) 1991-06-20 1996-07-23 Rh one-Poulenc Rorer Pharmaceuticals Inc. Therapeutic fragments of von Willebrand factor
US5567588A (en) 1990-06-11 1996-10-22 University Research Corporation Systematic evolution of ligands by exponential enrichment: Solution SELEX
US5580737A (en) 1990-06-11 1996-12-03 Nexstar Pharmaceuticals, Inc. High-affinity nucleic acid ligands that discriminate between theophylline and caffeine
US5614531A (en) 1994-02-19 1997-03-25 Merck Patent Gesellschaft Mit Beschrankter Haftung Adhesion receptor antagonists
US5627046A (en) 1991-08-21 1997-05-06 Rhone-Poulenc Rorer S.A. Yeast promoter and its use
US5635615A (en) 1990-06-11 1997-06-03 Nexstar Pharmaceuticals, Inc. High affinity HIV nucleocapsid nucleic acid ligands
US5637459A (en) 1990-06-11 1997-06-10 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: chimeric selex
US5648214A (en) 1990-06-11 1997-07-15 University Research Corporation High-affinity oligonucleotide ligands to the tachykinin substance P
US5654151A (en) 1990-06-11 1997-08-05 Nexstar Pharmaceuticals, Inc. High affinity HIV Nucleocapsid nucleic acid ligands
US5660985A (en) 1990-06-11 1997-08-26 Nexstar Pharmaceuticals, Inc. High affinity nucleic acid ligands containing modified nucleotides
US5663060A (en) 1992-04-07 1997-09-02 Emory University Hybrid human/animal factor VIII
US5668264A (en) 1990-06-11 1997-09-16 Nexstar Pharmaceuticals, Inc. High affinity PDGF nucleic acid ligands
US5674685A (en) 1990-06-11 1997-10-07 Nexstar Pharmaceuticals, Inc. High affinity PDGF nucleic acid ligands
US5683867A (en) 1990-06-11 1997-11-04 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: blended SELEX
US5686566A (en) 1989-06-16 1997-11-11 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5688912A (en) 1995-09-22 1997-11-18 Bayer Corporation Peptide ligands which bind to von willebrand factor
US5698687A (en) 1990-10-12 1997-12-16 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Modified ribozymls
US5705337A (en) 1990-06-11 1998-01-06 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: chemi-SELEX
US5707796A (en) 1990-06-11 1998-01-13 Nexstar Pharmaceuticals, Inc. Method for selecting nucleic acids on the basis of structure
US5710131A (en) 1993-07-01 1998-01-20 Merck Patent Gesellschaft Mit Beschrankter Haftung Inhibitor of collagen-stimulated platelet aggregation
US5763173A (en) 1990-06-11 1998-06-09 Nexstar Pharmaceuticals, Inc. Nucleic acid ligand inhibitors to DNA polymerases
US5763177A (en) 1990-06-11 1998-06-09 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: photoselection of nucleic acid ligands and solution selex
US5789157A (en) 1990-06-11 1998-08-04 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: tissue selex
US5817635A (en) 1993-08-09 1998-10-06 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Modified ribozymes
US5827516A (en) 1992-08-11 1998-10-27 President And Fellows Of Harvard College Immunomodulatory peptides
US5830880A (en) 1994-08-26 1998-11-03 Hoechst Aktiengesellschaft Gene therapy of tumors with an endothelial cell-specific, cell cycle-dependent active compound
US5837488A (en) 1990-03-02 1998-11-17 Bio-Technology General Corp. Cloning and production of human von Willebrand Factor GP1b binding domain polypeptides and methods of using same
US5837486A (en) 1988-12-22 1998-11-17 Genentech, Inc. Method for preparing soluble analogues of integrins
US5847086A (en) 1991-06-20 1998-12-08 Centeon L.L.C. Therapeutic fragments of von Willebrand factor
US5854005A (en) 1994-09-29 1998-12-29 Mount Sinai School Of Medicine Platelet blockade assay
US5854403A (en) 1994-10-04 1998-12-29 Immuno Aktiengesellschaft Method for isolation of highly pure von Willebrand Factor
US5856126A (en) 1993-09-22 1999-01-05 Ajinomoto Co., Inc. Peptide having anti-thrombus activity and method of producing the same
US5861254A (en) 1997-01-31 1999-01-19 Nexstar Pharmaceuticals, Inc. Flow cell SELEX
US5866113A (en) 1996-05-31 1999-02-02 Medtronic, Inc. Medical device with biomolecule-coated surface graft matrix
US5869615A (en) 1994-01-03 1999-02-09 Washington University Modified complement proteases
US5900476A (en) 1986-05-30 1999-05-04 The Scripps Research Institute Therapeutic domains of van Willebrand factor
US6008193A (en) 1990-03-02 1999-12-28 Bio-Technology General Corp. Methods of using human von Willebrand factor GPIb binding domain polypeptides
US6011020A (en) 1990-06-11 2000-01-04 Nexstar Pharmaceuticals, Inc. Nucleic acid ligand complexes
US6013443A (en) 1995-05-03 2000-01-11 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: tissue SELEX
US6020130A (en) 1995-06-07 2000-02-01 Nexstar Pharmaceuticals, Inc. Nucleic acid ligands that bind to and inhibit DNA polymerases
US6028090A (en) 1993-09-23 2000-02-22 Merck Patent Gesellschaft Mit Beschrankter Haftung Adhesion receptor antagonists III
US6033856A (en) 1997-03-14 2000-03-07 Hoechst Aktiengesellschaft Promoter of the cdc25B gene, its preparation and use
US6037329A (en) 1994-03-15 2000-03-14 Selective Genetics, Inc. Compositions containing nucleic acids and ligands for therapeutic treatment
US6040143A (en) 1996-07-19 2000-03-21 The Regents Of The University Of Michigan DNA encoding von Willebrand factor and methods of use
US6051698A (en) 1997-06-06 2000-04-18 Janjic; Nebojsa Vascular endothelial growth factor (VEGF) nucleic acid ligand complexes
US6068838A (en) 1996-04-29 2000-05-30 Baxter Aktiengesellschaft Purified multimerase
US6074832A (en) 1996-07-19 2000-06-13 The Regents Of The University Of Michigan DNA encoding canine von Willebrand factor and methods of use
US6100061A (en) 1997-06-20 2000-08-08 Immuno Aktiengesellschaft Recombinant cell clone having increased stability in serum- and protein-free medium and a method of recovering the stable cell clone and the production of recombinant proteins by using a stable cell clone
US6114135A (en) 1991-11-08 2000-09-05 Goldstein; Sheldon Multiple coagulation test system and method of using a multiple coagulation test system
US6177059B1 (en) 1996-02-07 2001-01-23 Yoshitomi Pharmaceutical Industries, Ltd. GPIb-lipid complex and uses thereof
US6207816B1 (en) 1995-06-02 2001-03-27 Nexstar Pharmaceuticals, Inc. High affinity oligonucleotide ligands to growth factors
US6210929B1 (en) 1995-11-24 2001-04-03 Baxter Aktiengesellschaft Fusion protein comprising a furin derivative or a derivative of a furin analogue and a heterologous sequence
US6221623B1 (en) 1997-11-10 2001-04-24 The Regents Of The University Of California Biochemical methods for detecting cervical dysplasia and cancer
US6229002B1 (en) 1995-06-07 2001-05-08 Nexstar Pharmaceuticlas, Inc. Platelet derived growth factor (PDGF) nucleic acid ligand complexes
US6239261B1 (en) 1989-02-14 2001-05-29 Aventis Behring Gmbh Pasteurized, purified von Willebrand factor concentrate and a process for the preparation thereof
US6255554B1 (en) 1994-09-21 2001-07-03 American National Red Cross Transgenic non-human mammals expressing human coagulation factor VIII and von Willebrand factor
US6280731B1 (en) 1994-11-30 2001-08-28 Ajinomoto Co., Inc. Antithrombotic agent and anti-von willebrand factor monoclonal antibody
US6320029B1 (en) 1996-11-29 2001-11-20 The American National Red Cross Methods of production and use of liquid formulations of plasma proteins
US6346614B1 (en) 1992-07-23 2002-02-12 Hybridon, Inc. Hybrid oligonucleotide phosphorothioates
US6410237B1 (en) 1997-07-18 2002-06-25 Board Of Trustees Operating Michigan State University DNA encoding canine von Willebrand factor and methods of use
US6451976B1 (en) 1997-03-20 2002-09-17 Trigen Limited Bi-or multifunctional molecules based on a dendroaspin scaffold
US6465624B1 (en) 1997-02-27 2002-10-15 Baxter Aktiengesellschaft Purification of von-Willebrand factor by cation exchanger chromatography
US6475725B1 (en) 1997-06-20 2002-11-05 Baxter Aktiengesellschaft Recombinant cell clones having increased stability and methods of making and using the same
US6489290B2 (en) 1997-03-27 2002-12-03 Trustees Of Boston University Antiplatelet agent
US6518482B2 (en) 1994-09-13 2003-02-11 American National Red Cross Transgenic non-human mammals expressing human coagulation factor VIII and von Willebrand factor
US6521594B1 (en) 1990-04-06 2003-02-18 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6576758B1 (en) 1996-09-24 2003-06-10 Aventis Pharma Deutschland Gmbh Nucleic acid constructs containing hybrid promoters
US6579723B1 (en) 1997-02-27 2003-06-17 Baxter Aktiengesellschaft Method of recovering highly purified vWF or factor VIII/vWF-complex
US6656731B1 (en) 1997-09-22 2003-12-02 Max Planck Gesellschaft Zur Forderung Der Wissenschaften E.V. Nucleic acid catalysts with endonuclease activity
US6686179B2 (en) 1992-01-31 2004-02-03 Aventis Behring L.L.C. Fusion polypeptides of human serum albumin and a therapeutically active polypeptide
USRE38431E1 (en) 1995-12-01 2004-02-17 The American National Red Cross Methods of production and use of liquid formulations of plasma proteins
WO2004015425A1 (en) 2002-08-07 2004-02-19 Umc Utrecht Holding B.V. Modulation of platelet adhesion based on the surface exposed beta-switch loop of platelet glycoprotein ib-alpha
US6737405B2 (en) 2000-05-08 2004-05-18 Aventis Behring Gmbh Stabilized protein preparation and process for its preparation
US6780583B1 (en) 1996-07-19 2004-08-24 Board Of Trustees Operating Michigan State University DNA encoding canine von willebrand factor and methods of use
US20040180360A1 (en) 2002-11-21 2004-09-16 Charles Wilson Multivalent aptamer therapeutics with improved pharmacodynamic properties and methods of making and using the same
US20040197804A1 (en) 2002-12-03 2004-10-07 Keefe Anthony D. Method for in vitro selection of 2'-substituted nucleic acids
US20050037394A1 (en) 2002-12-03 2005-02-17 Keefe Anthony D. Method for in vitro selection of 2'-substituted nucleic acids
US20050136056A1 (en) 2002-07-29 2005-06-23 Shunsuke Kageyama Pharmaceutical composition for the treatment of thrombocytopenia
US20060057573A1 (en) 2002-02-15 2006-03-16 Somalogic, Inc Methods and reagents for detecting target binding by nucleic acid ligands
US7566701B2 (en) * 2004-09-07 2009-07-28 Archemix Corp. Aptamers to von Willebrand Factor and their use as thrombotic disease therapeutics

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2548562B1 (fr) * 1983-07-08 1989-02-24 Commissariat Energie Atomique Lopin composite pour transformation a chaud
AU1435492A (en) * 1991-02-21 1992-09-15 Gilead Sciences, Inc. Aptamer specific for biomolecules and method of making
US5180853A (en) * 1992-05-04 1993-01-19 Dave Paritosh R 4,4-6,6-tetronitroadamantan-2-one
AU1838295A (en) * 1994-02-10 1995-08-29 Nexagen, Inc. High-affinity ligands of basic fibroblast growth factor and thrombin
US6228360B1 (en) * 1998-08-19 2001-05-08 Ajinomoto Co., Inc. Antithrombotic agent and humanized anti-von Willebrand factor monoclonal antibody

Patent Citations (138)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3710795A (en) 1970-09-29 1973-01-16 Alza Corp Drug-delivery device with stretched, rate-controlling membrane
US4959309A (en) 1983-07-14 1990-09-25 Molecular Diagnostics, Inc. Fast photochemical method of labelling nucleic acids for detection purposes in hybridization assays
US4666884A (en) 1984-04-10 1987-05-19 New England Deaconess Hospital Method of inhibiting binding of von Willebrand factor to human platelets and inducing interaction of platelets with vessel walls
GB2183661A (en) 1985-03-30 1987-06-10 Marc Ballivet Method for obtaining dna, rna, peptides, polypeptides or proteins by means of a dna recombinant technique
US5723323A (en) 1985-03-30 1998-03-03 Kauffman; Stuart Alan Method of identifying a stochastically-generated peptide, polypeptide, or protein having ligand binding property and compositions thereof
EP0197592B1 (en) 1985-04-01 1993-02-17 Stichting Vrienden Van De Stichting Dr. Karl Landsteiner Preparation of the human von willebrand factor by recombinant dna
US5180583A (en) 1985-11-26 1993-01-19 Hedner Ulla K E Method for the treatment of bleeding disorders
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683195B1 (es) 1986-01-30 1990-11-27 Cetus Corp
US5900476A (en) 1986-05-30 1999-05-04 The Scripps Research Institute Therapeutic domains of van Willebrand factor
US5238919A (en) 1986-05-30 1993-08-24 Scipps Clinic And Research Foundation Peptides that inhibit von Willebrand Factor binding to the platelet SPIB receptor
US4935363A (en) 1987-03-30 1990-06-19 Board Of Regents, The University Of Texas System Sterol regulatory elements
US5260274A (en) 1987-05-01 1993-11-09 Scripps Clinic And Research Foundation Factor VIII binding domain of von Willebrand Factor
US5260274B1 (en) 1987-05-01 1998-01-20 Scripps Research Inst Factor VIII binding domain of von willebrand factor
US5043429A (en) 1987-05-01 1991-08-27 Scripps Clinic And Research Foundation Protein fragments containing Factor VIII binding domain of von Willebrand Factor
US5597711A (en) 1987-05-01 1997-01-28 The Scripps Research Institute Factor VIII binding domain of von Willebrand factor
US5043429B1 (en) 1987-05-01 1997-10-14 Scripps Research Inst Protein fragments containing factor VIII binding domain of von willebrand factor
US5171844A (en) 1987-06-12 1992-12-15 Gist-Brocades N.W. Proteins with factor viii activity: process for their preparation using genetically-engineered cells and pharmaceutical compositions containing them
US5200510A (en) 1987-06-16 1993-04-06 Zymogenetics, Inc. Method for purifying factor viii:c, von willebrand factor and complexes thereof
US5112950A (en) 1987-08-11 1992-05-12 Transgene S.A. Factor viii analog, preparation process, and pharmaceutical composition containing it
US5340727A (en) 1987-11-17 1994-08-23 The Scripps Research Institute GPIbα fragments and recombinant DNA expression vectors
US5837486A (en) 1988-12-22 1998-11-17 Genentech, Inc. Method for preparing soluble analogues of integrins
US6239261B1 (en) 1989-02-14 2001-05-29 Aventis Behring Gmbh Pasteurized, purified von Willebrand factor concentrate and a process for the preparation thereof
US5686570A (en) 1989-06-16 1997-11-11 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5686567A (en) 1989-06-16 1997-11-11 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5686569A (en) 1989-06-16 1997-11-11 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5318899A (en) 1989-06-16 1994-06-07 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5686566A (en) 1989-06-16 1997-11-11 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5958732A (en) 1989-06-16 1999-09-28 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5686568A (en) 1989-06-16 1997-11-11 Cor Therapeutics, Inc. Platelet aggregration inhibitors
US5686571A (en) 1989-06-16 1997-11-11 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5070010A (en) 1989-10-30 1991-12-03 Hoffman-La Roche Inc. Method for determining anti-viral transactivating activity
US5849702A (en) 1990-03-02 1998-12-15 Bio-Technology General Corp. Cloning and production of human von Willebrand factor GPIB binding domain polypeptides and methods of using same
US5837488A (en) 1990-03-02 1998-11-17 Bio-Technology General Corp. Cloning and production of human von Willebrand Factor GP1b binding domain polypeptides and methods of using same
US5849536A (en) 1990-03-02 1998-12-15 Bio-Technology General Corp. Cloning and production of human von willebrand factor GPIb binding domain polypeptides and methods of using same
US6008193A (en) 1990-03-02 1999-12-28 Bio-Technology General Corp. Methods of using human von Willebrand factor GPIb binding domain polypeptides
US6521594B1 (en) 1990-04-06 2003-02-18 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US5660985A (en) 1990-06-11 1997-08-26 Nexstar Pharmaceuticals, Inc. High affinity nucleic acid ligands containing modified nucleotides
US5674685A (en) 1990-06-11 1997-10-07 Nexstar Pharmaceuticals, Inc. High affinity PDGF nucleic acid ligands
US5707796A (en) 1990-06-11 1998-01-13 Nexstar Pharmaceuticals, Inc. Method for selecting nucleic acids on the basis of structure
US5580737A (en) 1990-06-11 1996-12-03 Nexstar Pharmaceuticals, Inc. High-affinity nucleic acid ligands that discriminate between theophylline and caffeine
US5789157A (en) 1990-06-11 1998-08-04 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: tissue selex
US5763177A (en) 1990-06-11 1998-06-09 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: photoselection of nucleic acid ligands and solution selex
US5635615A (en) 1990-06-11 1997-06-03 Nexstar Pharmaceuticals, Inc. High affinity HIV nucleocapsid nucleic acid ligands
US5637459A (en) 1990-06-11 1997-06-10 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: chimeric selex
US5648214A (en) 1990-06-11 1997-07-15 University Research Corporation High-affinity oligonucleotide ligands to the tachykinin substance P
US5654151A (en) 1990-06-11 1997-08-05 Nexstar Pharmaceuticals, Inc. High affinity HIV Nucleocapsid nucleic acid ligands
US5503978A (en) 1990-06-11 1996-04-02 University Research Corporation Method for identification of high affinity DNA ligands of HIV-1 reverse transcriptase
US5763173A (en) 1990-06-11 1998-06-09 Nexstar Pharmaceuticals, Inc. Nucleic acid ligand inhibitors to DNA polymerases
US5668264A (en) 1990-06-11 1997-09-16 Nexstar Pharmaceuticals, Inc. High affinity PDGF nucleic acid ligands
US5475096A (en) 1990-06-11 1995-12-12 University Research Corporation Nucleic acid ligands
US5459015A (en) 1990-06-11 1995-10-17 Nexstar Pharmaceuticals, Inc. High-affinity RNA ligands of basic fibroblast growth factor
US5683867A (en) 1990-06-11 1997-11-04 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: blended SELEX
US5958691A (en) 1990-06-11 1999-09-28 Nexstar Pharmaceuticals, Inc. High affinity nucleic acid ligands containing modified nucleotides
US5705337A (en) 1990-06-11 1998-01-06 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: chemi-SELEX
US5567588A (en) 1990-06-11 1996-10-22 University Research Corporation Systematic evolution of ligands by exponential enrichment: Solution SELEX
US5496938A (en) 1990-06-11 1996-03-05 Nexstar Pharmaceuticals, Inc. Nucleic acid ligands to HIV-RT and HIV-1 rev
US6011020A (en) 1990-06-11 2000-01-04 Nexstar Pharmaceuticals, Inc. Nucleic acid ligand complexes
US5270163A (en) 1990-06-11 1993-12-14 University Research Corporation Methods for identifying nucleic acid ligands
US5698687A (en) 1990-10-12 1997-12-16 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Modified ribozymls
US5965425A (en) 1990-11-26 1999-10-12 Genetics Institute, Inc. Expression of pace in host cells and methods of use thereof
US5460950A (en) 1990-11-26 1995-10-24 Genetics Institute, Inc. Expression of PACE in host cells and methods of use thereof
US5321127A (en) 1991-03-18 1994-06-14 Brigham And Women's Hospital Antiplatelet and antithrombotic activity of platelet glycoprotein Ib receptor fragments
US5539086A (en) 1991-06-20 1996-07-23 Rh one-Poulenc Rorer Pharmaceuticals Inc. Therapeutic fragments of von Willebrand factor
US5847086A (en) 1991-06-20 1998-12-08 Centeon L.L.C. Therapeutic fragments of von Willebrand factor
US5627046A (en) 1991-08-21 1997-05-06 Rhone-Poulenc Rorer S.A. Yeast promoter and its use
US5593959A (en) 1991-10-07 1997-01-14 The Research Foundation Of State University Of New York Mutations in the gene encoding the alpha chain of platelet glycoprotein Ib
US5624817A (en) 1991-10-07 1997-04-29 The Research Foundation Of State University Of New York Mutations in the gene encoding the alpha chain of platelet glycoprotein Ib
US5317097A (en) 1991-10-07 1994-05-31 The Research Foundation Of State University Of New York Mutations in the gene encoding the α chain on platelet glycoprotein IB
US5298239A (en) 1991-10-07 1994-03-29 The Research Foundation Of State University Of New York Mutations rendering platelet glycoprotein IB α less reactive
US6114135A (en) 1991-11-08 2000-09-05 Goldstein; Sheldon Multiple coagulation test system and method of using a multiple coagulation test system
US5366869A (en) 1991-11-08 1994-11-22 Sheldon Goldstein Multiple coagulation test device and method
US5336667A (en) 1991-12-03 1994-08-09 Temple University Of The Commonwealth System Of Higher Education Method for inhibiting the ahesion of platelet with alboaggregins: platelet agonists which bind to platelet membrane glycoprotein IB
US6686179B2 (en) 1992-01-31 2004-02-03 Aventis Behring L.L.C. Fusion polypeptides of human serum albumin and a therapeutically active polypeptide
US5663060A (en) 1992-04-07 1997-09-02 Emory University Hybrid human/animal factor VIII
US5364771A (en) 1992-04-07 1994-11-15 Emory University Hybrid human/porcine factor VIII
US6346614B1 (en) 1992-07-23 2002-02-12 Hybridon, Inc. Hybrid oligonucleotide phosphorothioates
US5827516A (en) 1992-08-11 1998-10-27 President And Fellows Of Harvard College Immunomodulatory peptides
US5338671A (en) 1992-10-07 1994-08-16 Eastman Kodak Company DNA amplification with thermostable DNA polymerase and polymerase inhibiting antibody
EP0592035B1 (en) 1992-10-07 1996-01-03 Johnson & Johnson Clinical Diagnostics, Inc. Thermostable DNA polymerase composition comprising a temperature sensitive polymerase inhibitor, diagnostic test kits and methods of use
US5262564A (en) 1992-10-30 1993-11-16 Octamer, Inc. Sulfinic acid adducts of organo nitroso compounds useful as retroviral inactivating agents anti-retroviral agents and anti-tumor agents
US5532255A (en) 1993-05-01 1996-07-02 Merck Patent Gesellschaft Mit Beschrankter Haftung Adhesion receptor antagonists
US5710131A (en) 1993-07-01 1998-01-20 Merck Patent Gesellschaft Mit Beschrankter Haftung Inhibitor of collagen-stimulated platelet aggregation
US5817635A (en) 1993-08-09 1998-10-06 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Modified ribozymes
US5856126A (en) 1993-09-22 1999-01-05 Ajinomoto Co., Inc. Peptide having anti-thrombus activity and method of producing the same
US6028090A (en) 1993-09-23 2000-02-22 Merck Patent Gesellschaft Mit Beschrankter Haftung Adhesion receptor antagonists III
US5869615A (en) 1994-01-03 1999-02-09 Washington University Modified complement proteases
US5928892A (en) 1994-01-03 1999-07-27 Washington University Modified complement proteases
US5614531A (en) 1994-02-19 1997-03-25 Merck Patent Gesellschaft Mit Beschrankter Haftung Adhesion receptor antagonists
US6503886B1 (en) 1994-03-15 2003-01-07 Selective Genetics, Inc. Compositions containing nucleic acids and ligands for therapeutic treatment
US6037329A (en) 1994-03-15 2000-03-14 Selective Genetics, Inc. Compositions containing nucleic acids and ligands for therapeutic treatment
US5462752A (en) 1994-07-28 1995-10-31 Prp, Inc. Inhibition of platelet binding
US5830880A (en) 1994-08-26 1998-11-03 Hoechst Aktiengesellschaft Gene therapy of tumors with an endothelial cell-specific, cell cycle-dependent active compound
US6358732B1 (en) 1994-08-26 2002-03-19 Aventis Pharma Deutschland Gmbh DNA for expression under control of a cell cycle-dependent promoter
US6518482B2 (en) 1994-09-13 2003-02-11 American National Red Cross Transgenic non-human mammals expressing human coagulation factor VIII and von Willebrand factor
US6255554B1 (en) 1994-09-21 2001-07-03 American National Red Cross Transgenic non-human mammals expressing human coagulation factor VIII and von Willebrand factor
US5854005A (en) 1994-09-29 1998-12-29 Mount Sinai School Of Medicine Platelet blockade assay
US5854403A (en) 1994-10-04 1998-12-29 Immuno Aktiengesellschaft Method for isolation of highly pure von Willebrand Factor
US5880265A (en) 1994-10-04 1999-03-09 Immuno Aktiengesellschaft Method for isolation of highly pure von willebrand factor
US6103693A (en) 1994-10-04 2000-08-15 Immuno Aktiengesellschaft Method for isolation of highly pure von willebrand factor
US6793920B2 (en) 1994-11-19 2004-09-21 Ajinomoto Co., Inc. Antithrombotic agent and anti-von willebrand factor monoclonal antibody
US6280731B1 (en) 1994-11-30 2001-08-28 Ajinomoto Co., Inc. Antithrombotic agent and anti-von willebrand factor monoclonal antibody
US6013443A (en) 1995-05-03 2000-01-11 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: tissue SELEX
US6207816B1 (en) 1995-06-02 2001-03-27 Nexstar Pharmaceuticals, Inc. High affinity oligonucleotide ligands to growth factors
US6229002B1 (en) 1995-06-07 2001-05-08 Nexstar Pharmaceuticlas, Inc. Platelet derived growth factor (PDGF) nucleic acid ligand complexes
US6020130A (en) 1995-06-07 2000-02-01 Nexstar Pharmaceuticals, Inc. Nucleic acid ligands that bind to and inhibit DNA polymerases
US5688912A (en) 1995-09-22 1997-11-18 Bayer Corporation Peptide ligands which bind to von willebrand factor
US6210929B1 (en) 1995-11-24 2001-04-03 Baxter Aktiengesellschaft Fusion protein comprising a furin derivative or a derivative of a furin analogue and a heterologous sequence
USRE38431E1 (en) 1995-12-01 2004-02-17 The American National Red Cross Methods of production and use of liquid formulations of plasma proteins
US6177059B1 (en) 1996-02-07 2001-01-23 Yoshitomi Pharmaceutical Industries, Ltd. GPIb-lipid complex and uses thereof
US6068838A (en) 1996-04-29 2000-05-30 Baxter Aktiengesellschaft Purified multimerase
US5866113A (en) 1996-05-31 1999-02-02 Medtronic, Inc. Medical device with biomolecule-coated surface graft matrix
US6074832A (en) 1996-07-19 2000-06-13 The Regents Of The University Of Michigan DNA encoding canine von Willebrand factor and methods of use
US6780583B1 (en) 1996-07-19 2004-08-24 Board Of Trustees Operating Michigan State University DNA encoding canine von willebrand factor and methods of use
US6040143A (en) 1996-07-19 2000-03-21 The Regents Of The University Of Michigan DNA encoding von Willebrand factor and methods of use
US6576758B1 (en) 1996-09-24 2003-06-10 Aventis Pharma Deutschland Gmbh Nucleic acid constructs containing hybrid promoters
US6320029B1 (en) 1996-11-29 2001-11-20 The American National Red Cross Methods of production and use of liquid formulations of plasma proteins
US5861254A (en) 1997-01-31 1999-01-19 Nexstar Pharmaceuticals, Inc. Flow cell SELEX
US6579723B1 (en) 1997-02-27 2003-06-17 Baxter Aktiengesellschaft Method of recovering highly purified vWF or factor VIII/vWF-complex
US6465624B1 (en) 1997-02-27 2002-10-15 Baxter Aktiengesellschaft Purification of von-Willebrand factor by cation exchanger chromatography
US6033856A (en) 1997-03-14 2000-03-07 Hoechst Aktiengesellschaft Promoter of the cdc25B gene, its preparation and use
US6451976B1 (en) 1997-03-20 2002-09-17 Trigen Limited Bi-or multifunctional molecules based on a dendroaspin scaffold
US6489290B2 (en) 1997-03-27 2002-12-03 Trustees Of Boston University Antiplatelet agent
US6051698A (en) 1997-06-06 2000-04-18 Janjic; Nebojsa Vascular endothelial growth factor (VEGF) nucleic acid ligand complexes
US6100061A (en) 1997-06-20 2000-08-08 Immuno Aktiengesellschaft Recombinant cell clone having increased stability in serum- and protein-free medium and a method of recovering the stable cell clone and the production of recombinant proteins by using a stable cell clone
US6475725B1 (en) 1997-06-20 2002-11-05 Baxter Aktiengesellschaft Recombinant cell clones having increased stability and methods of making and using the same
US6410237B1 (en) 1997-07-18 2002-06-25 Board Of Trustees Operating Michigan State University DNA encoding canine von Willebrand factor and methods of use
US6656731B1 (en) 1997-09-22 2003-12-02 Max Planck Gesellschaft Zur Forderung Der Wissenschaften E.V. Nucleic acid catalysts with endonuclease activity
US6221623B1 (en) 1997-11-10 2001-04-24 The Regents Of The University Of California Biochemical methods for detecting cervical dysplasia and cancer
US6737405B2 (en) 2000-05-08 2004-05-18 Aventis Behring Gmbh Stabilized protein preparation and process for its preparation
US20060057573A1 (en) 2002-02-15 2006-03-16 Somalogic, Inc Methods and reagents for detecting target binding by nucleic acid ligands
US20050136056A1 (en) 2002-07-29 2005-06-23 Shunsuke Kageyama Pharmaceutical composition for the treatment of thrombocytopenia
WO2004015425A1 (en) 2002-08-07 2004-02-19 Umc Utrecht Holding B.V. Modulation of platelet adhesion based on the surface exposed beta-switch loop of platelet glycoprotein ib-alpha
US20050192224A1 (en) 2002-08-07 2005-09-01 Huizinga Eric G. Modulation platelet adhesion based on the surface-exposed beta-switch loop of platelet glycoprotein IB-alpha
US20040180360A1 (en) 2002-11-21 2004-09-16 Charles Wilson Multivalent aptamer therapeutics with improved pharmacodynamic properties and methods of making and using the same
US20040197804A1 (en) 2002-12-03 2004-10-07 Keefe Anthony D. Method for in vitro selection of 2'-substituted nucleic acids
US20050037394A1 (en) 2002-12-03 2005-02-17 Keefe Anthony D. Method for in vitro selection of 2'-substituted nucleic acids
US7566701B2 (en) * 2004-09-07 2009-07-28 Archemix Corp. Aptamers to von Willebrand Factor and their use as thrombotic disease therapeutics

Non-Patent Citations (103)

* Cited by examiner, † Cited by third party
Title
Altschul et al., "Basic Local Alignment Search Tool", (1990) J. Mol. Biol. 215: 403-410.
Altschul et al., "Gapped BLAST and PSI-BLAST: A New Generation of Protein Database Search Programs", (1997) Nuc. Acids Res. 15: 3389-3402.
Andrake, "DNA polymerase of bacteriophage T4 is an autogenous translational repressor ", (1988) Proc. Natl. Acad. Sci. USA 85: 7942-7946.
Barabino et al , "Inhibition of Sickle Erythrocyte Adhesion to Immobilized Thrombospondin by von Willebrand Factor Under Dynamic Flow Conditions", (1997) Blood 89: 2560-2567.
Bonnefoy et al , "Inhibition of von Willebrand factor-GP1b/IX/V interactions as a straegy to prevent arterial thrombosis", (2003) Exp. Rev. Cario. Ther., Future Drugs 1(2):257-269.
Burmeister et al., "Direct in vitro Selection of a 2′-O-Methyl Aptamer to VEGF", (2005) Chemistry and Biology 12: 25-33.
Burmeister et al., "Direct in vitro Selection of a 2'-O-Methyl Aptamer to VEGF", (2005) Chemistry and Biology 12: 25-33.
Carey et al., "Sequence-specific interaction of R17 coat protein with its ribonucleic acid binding site", (1983) Biochem. 22: 2601-2610.
Chelliserrykatil and Ellington, "Evolution of a T7 RNA Polymerase Variant That Transcribes 2′-O-Methyl RNA", (2004) Nat. Biotech 9: 1150-1160.
Chelliserrykatil and Ellington, "Evolution of a T7 RNA Polymerase Variant That Transcribes 2'-O-Methyl RNA", (2004) Nat. Biotech 9: 1150-1160.
Chen et al., "Selection of high-affinity RNA ligands to reverse transcriptase. Inhibition of cDNA synthesis and RNase H activity", (1994) Biochem. 33: 8746-8756.
Cohen et al., "Interactions of hormonal steroids with nucleic acids: A specific requirement for guanine", (1969) Proc. Natl. Acad. Sci. USA 63: 458-464.
Cotton et al., "2′-O-methyl, 2′-O-ethyl oligoribonucleotides and phosphorothioate oligodeoxyribonucleotides as inhibitors of the in vitro U7 snRNP-dependent mRNA processing event", (1991) Nucleic Acids Res. 19: 2629-2635.
Cotton et al., "2'-O-methyl, 2'-O-ethyl oligoribonucleotides and phosphorothioate oligodeoxyribonucleotides as inhibitors of the in vitro U7 snRNP-dependent mRNA processing event", (1991) Nucleic Acids Res. 19: 2629-2635.
Davis et al., "Identifying consensus patterns and secondary structure in SELEX sequence sets", (1996) Methods in Enzymology 267: 302-314.
Dehouck et al., "Blood-brain barrier in vitro-rapid evaluation of strategies for achieving drug targeting to the central nervous system", (1996) Biol. Physiology of the Blood Brain Barrier, Couraud and Scherman eds., 23: 143-146.
Ellington and Szostak, "Abstracts of papers presented at the 1990 meeting on RNA Processing", (1990) Cold Spring Harbor, NY, p. 84.
Fitzwater et al., "A SELEX primer", (1996) Methods in Enzymology 267: 275-301.
Froehler et al., "Deoxynucleoside H-Phosphonate Diester Intermediates in the Synthesis of Internucleotide Phosphate Analogues", (1986) Tet. Lett. 27: 5575-5578.
Froehler et al., "Synthesis of DNA via Deoxynucleoside H-phosphonate Intermediates", (1986) Nuc. Acids Res. 14: 5399-5407.
Gath et al., "The blood-CSF barrier in vitro", Biol. and Physiology of the Blood Brain Barrier, (1996) Couraud and Scherman eds., 25: 153-158.
Gold et al., "Diversity of Oligonucleotide Functions", (1995) Annu. Rev. Biochem. 64: 763-797.
Gralnick et al., "A monomeric von Willebrand factor fragment, Leu-504-Ser-728, inhibits von Willebrand factor interaction with glycoprotein Ib-IX", (1992) Proc. Natl. Acad. Sci. 89: 488-94.
Gray et al., "Rapid Measurement of Modified Oligonucleotide Levels in Plasma Samples With A Fluorophore Specific for Single-Stranded DNA", (1997) Antisense and Nucleic Acid Drug Development 7(3): 133-140.
Greenwald et al., "Highly Water Soluble Taxol Derivatives: 7-Polyethylene Glycol Carbamates and Carbonates", (1995) J. Org. Chem. 60: 331-336.
Harris et al., "Effect of Pegylation on Pharmaceuticals", (2003) Nature 2: 214-221.
Harrison et al., "The PFA-100: A potential rapid screening tool for the assessment of platelet dysfunction", (2002) Clin. Lab. Haem. 24: 225-232.
Hirose et al., "Rapid Synthesis of Trideoxyribonucleotide Blocks", (1978) Tet. Lett. 28: 2449-2452.
Hobbs et al., "Polynucleotides containing 2′-amino-2′-deoxyribose and 2′-azido-2′-deoxyribose", (1973) Biochemistry 12: 5138-5145.
Hobbs et al., "Polynucleotides containing 2'-amino-2'-deoxyribose and 2'-azido-2'-deoxyribose", (1973) Biochemistry 12: 5138-5145.
Huizinga et al., "Structures of glycoprotein 1b-alpha and its complex with von Willebrand factor A1 domain", (2002) Science 297: 1176-1179.
Hwang et al., "Inhibition of gene expression in human cells through small molecule-RNA interactions", (1999) PNAS 96(23):12997-13002.
Ikram et al., "An Experimental Model for the In-Vivo Study of Coronary Arterial Thrombosis", (1975) Angiology 26(4): 356-364.
Jackson and Schoenwaelder, "Antiplatelet Therapy: In Search of the Magic Bullet", (2003) Nature Reviews 2: 1-15.
Joyce and Inoue, "A novel technique for the rapid preparation of mutant RNAs", (1998) Nuc. Acids Res. 17: 711-722.
Joyce, "Amplification, mutation and selection of catalytic RNA", (1989) RNA: Catalysis, Splicing, Evolution, Amsterdam pp. 83-87.
Kacian et al., "A replicating RNA molecule suitable for a detailed analysis of extracellular evolution and replication", (1972) Proc. Natl. Acad. Sci. USA 69: 3038-3042.
Kadonaga et al., "Affinity purification of sequence-specific DNA binding proteins", (1986) Proc. Natl. Acad. Sci. USA 83: 5889-5893.
Kageyama et al., "Anti-Human von Willebrand Factor Monoclonal Antibody AJvW-2 Prevents Thrombus Deposition and Neointima Formation After Balloon Injury in Guinea Pigs", (2000) Arterioscler. Thromb. Vasc. Biol. 20: 2303-2308.
Kageyama et al., "Anti-Human vWF Monoclonal Antibody, AJvW-2 Fab, Inhibits Repetitive Coronary Artery Thrombosis without Bleeding Time Prolongation in Dogs", (2001) Thromb. Res. 101: 395-404.
Kageyama et al., "Anti-thrombotic effects and bleeding risk of AJvW-2, a monoclonal antibody against human von Willebrand factor", (1997) Br. J. Pharmacol. 122: 165-171.
Kageyama et al., "Effect of a humanized monoclonal antibody to von Willebrand factor in a canine model of coronary arterial thrombosis", (2002) Eur. J. Pharmacol. 443: 143-149.
Kageyama et al., "Pharmacokinetics and Pharmacodynamics of AJW200, a Humanized Monoclonal Antibody to von Willebrand Factor in Monkeys", (2002) Arterioscler Thromb. Vasc. Biol. pp. 187-192.
Kellogg et al., "Taqstart antibody(TM): 'Hot start' PCR facilitated by a neutralizing monoclonal antibody directed against taq DNA polymerase", (1994) BioTechniques 16(6): 1134-1137.
Kellogg et al., "Taqstart antibody™: ‘Hot start’ PCR facilitated by a neutralizing monoclonal antibody directed against taq DNA polymerase", (1994) BioTechniques 16(6): 1134-1137.
Kinzler and Vogelstein, "The GLI gene encodes a nuclear protein which binds specific sequences in the human genome", (1989) Mol. Cell. Biol. 10: 634-642.
Kinzler and Vogelstein, "Whole genome PCR: Application to the identification of sequences bound by gene regulatory proteins", (1989) Nuc. Acids Res. 17: 3645-3653.
Koppelman et al., "Requirements of von Willebrand Factor to Protect Factor VII From Inactivation by Activated Protein C", (1996) Blood 87: 2292-2300.
Kramer et al., "Evolution in vitro: Sequence and phenotype of a mutant RNA resistant to ethidium bromide", (1974) J. Mol. Biol. 89: 719-736.
Lestienne et al., "Inhibition of human leucocyte elastase by polynucleotides", (1983) Biochimie 65: 49-52.
Levisohn and Spiegelman, "Further extracellular Darwinian experiments with replicating RNA molecules: diverse variants isolated under different selective conditions", (1969) PNAS USA 63: 805-811.
Levisohn and Spiegelman, "The cloning of a self-replicating RNA molecule", (1968) PNAS USA 60: 866-872.
Ma and Ptashne, "A new class of yeast transcriptional activators", (1987) Cell 51: 113-119.
Maniatis et al., "Molecular Cloning: A Laboratory Manual", (1982) Cold Spring Harbor, NY p. 118.
Maniatis et al.,"Regulation of inducible and tissue-specific gene expression", (1987) Science 236: 1237-1245.
Mathews et al., "Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure", (2004) Proc. Nat. Acad. Sci. 101: 7287-7292.
McGhie et al., "Abolition of Cyclic Flow Variations in Stenosed, Endothelium-Injured Coronary Arteries in Nonhuman Primates with a Peptide Fragment (VCL) Derived from Human Plasma von Willebrand Factor-Glycoprotein Ib Binding Domain", (1994) Circulation 90(6): 2976-2981.
McGinnis et al., "BLAST: at the core of a powerful and diverse set of sequence analysis tools", (2004) Nuc. Acids Res. 32: W20-W25.
Miele et al., "Autocatalytic replication of a recombinant RNA", (1983) J. Mol. Biol. 171: 281-295.
Mills et al., "An extracellular Darwinian experiment with a self-duplicating nucleic acid molecule", (1967) Proc. Natl. Acad. Sci. USA 58: 217-220.
Mills et al., "Complete nucleotide sequence of a replicating RNA molecule", (1973) Science 180: 916-927.
Min et al., "Search for the optimal sequence of the ribosome binding site by random oligonucleotide-directed mutagenisis", (1988) Nuc. Acids Res. 16: 5075-5088.
Muesing et al., "Nucleic acid structure and expression of the human AIDS/lymphadenopathy retrovirus", (1985) Nature 313: 450-458.
Nimjee et al., "The potential of aptamers as anticoagulants", (2005) Trends Cardio Med. 15(1):41-45.
Oliphant and Struhl, "Defining the consensus sequences of E. coli promoter elements by random selection", (1988) Nuc. Acids Res.16: 7673-7683.
Oliphant and Struhl, "The use of random-sequence oligonucleotides for determining consensus sequences", (1987) Methods in Enzymology 155: 568-582.
Oliphant et al., "Cloning of random-sequence oligodeoxynucleotides", (1986) Gene 44: 177-183.
Oliphant et al., "Defining the sequence specificity of DNA-binding proteins by selecting binding sites from random-sequence oligonucleotides: analysis of yeast GCN4 protein", (1989) Mol. Cell. Biol. 9: 2944-2949.
Orgel, "Selection in vitro", (1979) Proc. R. Soc. Lon. B205: 435-442.
Ou et al., "DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells", (1988) Science 239: 295-297.
Padilla et al., "A Y639F/H784A T7 RNA polymerase double mutant displays superior properties for synthesizing RNAs with non-canonical NTPs", (2002) Nuc. Acids Res. 30: e138 (4 pgs).
Padilla et al., "Efficient synthesis of nucleic acids heavily modified with non-canonical ribose 2′-groups using a mutant T7 RNA polymerase (RNAP)", (1999) Nuc. Acids Res. 27(6): 1561-1563.
Padilla et al., "Efficient synthesis of nucleic acids heavily modified with non-canonical ribose 2'-groups using a mutant T7 RNA polymerase (RNAP)", (1999) Nuc. Acids Res. 27(6): 1561-1563.
Phillips and Scarborough, "Clinical Pharmacology of Eptifibatide", (1997) Am. J. Cardiol. 80(4A): 11B-20B.
Pietersz et al., "A 16-mer peptide (RQIKIWFQNRRMKWKK) from Antennapedia Preferentially Targets the Class I Pathway", (2001) Vaccine 19(11-12): 1397-1405.
Roberts et al., "Chemistry for peptide and protein PEGylation", (2002) Advanced Drug Delivery Rev. 54: 459-476.
Robertson and Joyce, "Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA", (1990) Nature 344: 467-468.
Romaniuk et al., "RNA binding site of R17 coat protein", (1987) Biochem. 26: 1563-1568.
Rote et al., "Chimeric 7E3 Prevents Carotid Artery Thrombosis in Cynomolgus Monkeys", (1994) Stroke 25: 1223-1233.
Rothbard et al. "Arginine-Rich Molecular Transporters for Drug Delivery: Role of Backbone Spacing in Cellular Uptake" (2002) J. Med. Chem. 45(17): 3612-3618.
Rothbard et al., "Conjugation of Arginine Oligomers to Cyclosporin A FacilitatesTopical Delivery and Inhibition of Inflammation", (2000) Nat. Med. 6(11): 1253-1257.
Ruckman et al., "2′-Fluoropyrimidine RNA-based Aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165): Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain", (1998) J. Biol. Chem. 273: 20556-20567-20695.
Ruckman et al., "2'-Fluoropyrimidine RNA-based Aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165): Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain", (1998) J. Biol. Chem. 273: 20556-20567-20695.
Rusconi et al., "RNA aptamers as reversible antagonists of coagulation factor Ixa", (2002) Nature 419:90-94.
Saffhill et al., "In vitro selection of bacteriophage Qbeta ribonucleic acid variants resistant to ethidium bromide", (1970) J. Mol. Biol. 51: 531-539.
Saffhill et al., "In vitro selection of bacteriophage Qβ ribonucleic acid variants resistant to ethidium bromide", (1970) J. Mol. Biol. 51: 531-539.
Sambrook et al., "Molecular Cloning: A Laboratory Manual", (1989) Cold Spring Harbor, NY, pp. 8.9-8.10.
Singleton et al., "Dictionary of Microbiol. and Molecular Biology", Wiley & Sons, New York, NY, 2nd ed. p. 493 (1991).
Sood et al., "A rapid and convenient synthesis of poly-thymidylic acid by the modified triester approach", (1977) Nuc. Acids Res. 4: 2757-2765.
Sproat et al., "New synthetic routes to synthons suitable for 2′-O-allyloligoribonucleotide assembly", (1991) Nuc. Acids Res. 19: 733-738.
Sproat et al., "New synthetic routes to synthons suitable for 2'-O-allyloligoribonucleotide assembly", (1991) Nuc. Acids Res. 19: 733-738.
Szostak, J.W., "Structure and Activity of Ribozymes" in "Redesigning the Molecules of Life" (1988) Springer-Verlag Berline Heidelberg, pp. 87-113.
Tanchou et al., "Monoclonal antibody-mediated inhibition of RNA binding and annealing activities of HIV type 1 nucleocapsid protein", (1994) Aids Research and Human Retroviruses 10: 983-993.
Thiesen and Bach, "Target detection assay (TDA): A versatile procedure to determine DNA binding sites as demonstrated on SPI protein", (1990) Nuc. Acids Res. 18: 3203-3209.
Uhlenbeck et al., "Interaction of R17 coat protein with its RNA binding site for translational repression", (1983) J. Biomolecular Structure and Dynamics 1: 539-552.
Vanhoorelbeke et al , "Inhibition of Platelet Adhesion to Collagen as a New Target for Antithrombotic Drugs", (2003) Cardio. Haema. Disorders 3: 125-140.
Vanhoorelbeke et al., "The role of VWF-collagen interaction in acute platelet thrombus formation", (2003) Drugs of the Future 28(1): 61-67.
Varughese et al., "Structure and Function of the Von Willebrand Factor A1 Domain", (2002) Current Protein and Peptide Science 3: 301-312.
Vives et al., "A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus", (1997) J. Biol. Chem. 27: 16010-16017.
Watson et al., "Molecular Biology of the Gene", (1987) Benjamin/Cummings Publishing Co., Inc. California, pp. 267, 295, 323, 361, 394, 396, 397 and 405.
White et al., "Developing aptamers into therapeutics", (2000) J. Clin. Invest. 106(8):929-934.
Wu et al , "Inhibition of the von Willebrand (VWF)-collagen interaction by an antihuman VWF monoclonal antibody results in abolition of in vivo arterial platelet thrombus formation in baboons", (2002) Blood 99(10): 3623-3628.
Yao et al., "Blockade of Platelet Membrane Glycoprotein Ib Receptors Delays Intracoronary Thrombogenesis, eEnhances Thrombolysis, and Delays Coronary Artery Reocclusion in Dogs", (1994) Circulation 89(6): 2822-2828.

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10695362B2 (en) 2010-05-14 2020-06-30 Tagcyx Biotechnologies Inc. Stabilization method of functional nucleic acid
WO2015117201A1 (en) * 2014-02-05 2015-08-13 Deakin University Aptamer construct
CN106164293A (zh) * 2014-02-05 2016-11-23 迪肯大学 适配子构建体
US10221420B2 (en) 2014-02-05 2019-03-05 Deakin University Aptamer construct
CN106164293B (zh) * 2014-02-05 2020-03-27 迪肯大学 适配子构建体
US10577610B2 (en) 2015-02-11 2020-03-03 Deakin University EpCAM aptamers and conjugates thereof

Also Published As

Publication number Publication date
AU2005287273A1 (en) 2006-03-30
WO2006033854A2 (en) 2006-03-30
US20090149643A1 (en) 2009-06-11
MX2007002772A (es) 2008-03-05
WO2006033854A3 (en) 2007-05-31
EP1789096A2 (en) 2007-05-30
EP1789096A4 (en) 2009-07-08
IL181689A0 (en) 2007-07-04
JP2008512098A (ja) 2008-04-24
AU2005287273B2 (en) 2011-05-12
SG156618A1 (en) 2009-11-26
BRPI0514984A (pt) 2008-07-01
KR20070101227A (ko) 2007-10-16
CA2579374A1 (en) 2006-03-30

Similar Documents

Publication Publication Date Title
US7589073B2 (en) Aptamers to von Willebrand Factor and their use as thrombotic disease therapeutics
US7998940B2 (en) Aptamers to von Willebrand factor and their use as thrombotic disease therapeutics
US7566701B2 (en) Aptamers to von Willebrand Factor and their use as thrombotic disease therapeutics
EP1991275B1 (en) Complement binding aptamers and anti-c5 agents useful in the treatment of ocular disorders
EP2578683B1 (en) Aptamer therapeutics useful in the treatment of complement-related disorders
US7998939B2 (en) Aptamers that bind thrombin with high affinity
AU2006214437C1 (en) Aptamer therapeutics useful in the treatment of complement-related disorders
US7579450B2 (en) Nucleic acid ligands specific to immunoglobulin E and their use as atopic disease therapeutics
WO2005052121A2 (en) Multivalent aptamers
AU2005245793A1 (en) Nucleic acid ligands specific to immunoglobulin e and their use as atopic disease therapeutics
US20090018093A1 (en) Nucleic Acid Ligands Specific to Immunoglobuline E and Their Use as Atopic Disease Therapeutics
AU2014218429B2 (en) Aptamer Therapeutics Useful in the Treatment of Complement-Related Disorders

Legal Events

Date Code Title Description
ZAAA Notice of allowance and fees due

Free format text: ORIGINAL CODE: NOA

ZAAB Notice of allowance mailed

Free format text: ORIGINAL CODE: MN/=.

AS Assignment

Owner name: ARCHEMIX CORP., MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:DIENER, JOHN L.;LAGASSE, H.A. DANIEL;BENEDICT, CLAUDE;SIGNING DATES FROM 20060329 TO 20060405;REEL/FRAME:026076/0348

STCF Information on status: patent grant

Free format text: PATENTED CASE

FPAY Fee payment

Year of fee payment: 4

MAFP Maintenance fee payment

Free format text: PAYMENT OF MAINTENANCE FEE, 8TH YEAR, LARGE ENTITY (ORIGINAL EVENT CODE: M1552); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY

Year of fee payment: 8

FEPP Fee payment procedure

Free format text: MAINTENANCE FEE REMINDER MAILED (ORIGINAL EVENT CODE: REM.); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY

LAPS Lapse for failure to pay maintenance fees

Free format text: PATENT EXPIRED FOR FAILURE TO PAY MAINTENANCE FEES (ORIGINAL EVENT CODE: EXP.); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY

STCH Information on status: patent discontinuation

Free format text: PATENT EXPIRED DUE TO NONPAYMENT OF MAINTENANCE FEES UNDER 37 CFR 1.362

FP Lapsed due to failure to pay maintenance fee

Effective date: 20230816